Raman Microscopy: Developments and Applications

Author: Turrel G. | Corset J.

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Raman Microscopy Developments and Applications Copyright © 1996 Elsevier Ltd. All rights reserved Shortcut URL to this page: http://www.sciencedirect.com/science/book/9780121896904 Edited by: George Turrell and Jacques Corset ISBN: 978-0-12-189690-4

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List of Contributors, Pages xv-xvi

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PDF (70 K) Preface, Pages xvii-xxii, Edgar S. Etz PDF (330 K) Acknowledgements, Pages xxiii-xxviii PDF (272 K) 1 - The Raman Effect, Pages 1-25, George Turrell Abstract | Abstract + References | PDF (1194 K) 2 - Characteristics of Raman Microscopy, Pages 27-49, George Turrell, Michel Delhaye and Paul Dhamelincourt Abstract | Abstract + References | PDF (848 K) 3 - Instrumentation, Pages 51-173, Michel Delhaye, Jacques Barbillat, Jean Aubard, Michel Bridoux and Edouard Da Silva Abstract | Abstract + References | PDF (5081 K) 4 - Raman Imaging, Pages 175-200, Jacques Barbillat Abstract | Abstract + References | PDF (1456 K) 5 - Raman Microscopy and Other Local Analysis Techniques, Pages 201-242, Michel Truchet, Jean-Claude Merlin and George Turrell Abstract | Abstract + References | PDF (1877 K) 6 - Application to Materials Science, Pages 243-287, Paul Dhamelincourt and Shin-ichi Nakashima Abstract | Abstract + References | PDF (1924 K) 7 - Applications in Earth, Planetary and Environmental Sciences, Pages 289-365, Paul F. McMillan, Jean Dubessy and Russell Hemley Abstract | Abstract + References | PDF (4079 K) 8 - Biological Applications, Pages 367-377, Michel Truchet Abstract | Abstract + References | PDF (620 K) 9 - Applications in Medicine, Pages 379-420, Michel Manfait and Igor Nabiev Abstract | Abstract + References | PDF (2303 K) 10 - Applications in Art, Jewelry and Forensic Science, Pages 421-453, Claude Coupry and Didier Brissaud Abstract | Abstract + References | PDF (1413 K) Index, Pages 455-463 PDF (342 K)

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List of Contributors

Jean Aubard, Institut de Topologie et de Dynamique des Systemes (CNRS URA 34), 1, rue Guy de la Brosse, 75005 Paris, France Jacques Barbillat, Laboratoire de Spectrochimie Infrarouge et Raman (CNRS UPR A2631L), Universite des Sciences et Technologic de Lille, 59655 Villeneuve d'Ascq, France Michel Bridoux, Laboratoire de Spectrochimie Infrarouge et Raman (CNRS UPR A2631L), Universite des Sciences et Technologic de Lille, 59655 Villeneuve d'Ascq, France Didier Brissaud, Laboratoire de Police Scientifique, 3, quai de I'Horloge, 75001 Paris, France Claude Coupry, Laboratoire de Spectrochimie Infrarouge et Raman (CNRS UPR A2631T), 2, rue Henri Dunant, 94320 Thiais, France Edouard Da Silva, DILOR, 255 ter, rue des Bois Blancs, 59000 Lille, France Michel Delhaye, DILOR, 255 ter, rue des Bois Blancs, 59000 Lille, France, Emeritus Professor, Universite des Sciencies et Technologic de Lille, 59655 Villeneuve d'Ascq, France Paul Dhamelincourt, Laboratoire de Spectrochimie Infrarouge et Raman (CNRS UPR A2631L), Universite des Sciences et Technologic de Lille, 59655 Villeneuve d'Ascq, France Jean Dubessy, CREGU (GDR CNRS 077). BP-23, 54501 Vandoeuvre-lesNancy, France Edgar S. Etz, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA Russell Hemley, Geophysical Laboratory (CIW), 5251 Broad Branch Road, N.W., Washington, DC 20015, USA Michel Manfait, Laboratoire de Spectroscopic Biomoleculaire, UFR de Pharmacie, Universite de Reims, 51, rue Cognacq-Jay, 51096 Reims, France Paul F. McMillan, Department of Chemistry, Arizona State University, Tempe, AZ 85287, USA

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List of Contributors

Jean-Claude Merlin, Laboratoire de Spectrochimie Infrarouge et Raman (CNRS UPR A2631L), Universite des Sciences et Technologic de Lille, 59655 Villeneuve d'Ascq, France Igor Nabiev, Laboratoire de Spectroscopic Biomoleculaire, UFR de Pharmacie, Universite de Reims, 51, rue Cognacq-Jay, 51096 Reims, France Shin-ichi Nakashima, Department of Applied Physics, Osaka University, Osaka, Japan Michel Truchet, Laboratoire d'Histophisiologie Fondamentale, Universite Pierre et Marie Curie, 12, rue Cuvier, 75005 Paris, France George Turrell, Laboratoire de Spectrochimie Infrarouge et Raman (CNRS UPR A2631L), Universite des Sciences et Technologic de Lille, 59655 Villeneuve d'Ascq, France

Preface

It is no surprise to see the micro-Raman Group at Lille come forth with this timely publication to document the present state of Raman microscopy. A quarter century has passed since the early attempts at Raman microsampling when the field began to merge with, and complement, other microprobe techniques. In the late 1960s to the early '70s, it was mainly the electron beam methods that opened up the microscopic domain to instrumental analysis, aside from classical light microscopy. In this realm, the principal goal was to obtain morphological, structural, and compositional information from the analyzed specimen. Scanning electron microscopes (SEMs), electron microprobes for x-ray microanalysis (EPMA), and analytical electron microscopes (AEMs) furnished detailed images of the sample and elemental compositional data from microscopic sampling volumes, for nearly all of the elements in the periodic table. Yet, at that time, one important piece of information was not available from any of these methodologies: the ability to link the compositional data to the atomic or molecular bonding of the elements, their speciation, such as structural coordination and stoichiometry, as well as crystallographic and amorphous structure. This analytical need for spatially resolved information on structure and bonding of the constituent elements brought forth the development of vibrational microspectroscopy. Infrared spectroscopy, of the non-Fourier transform (FT) variety, was widely used at the time, but infrared microspectroscopy was to fully emerge only in the late 1970s with the increasing use of FT-infrared instrumentation. Since the early 1960s, Raman spectroscopy had experienced a renaissance with the advent of the laser as the ideal excitation source. Laser radiation, from the near-ultraviolet across the visible spectrum, could be focused to the optical diffraction limit, for probe spots competitive with electron probing. Thus, it was then recognized that laser excitation utilizing optimally designed fore-optics and coupled to Raman instrumentation employing various types of sensitive detectors, would make possible Raman microspectroscopy and microscopy. This concept, and its earliest implementation, initiated the new frontiers of molecular Raman probing and imaging, to complement the elemental microprobe techniques with their imaging variants.

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The Laboratory for Infrared and Raman Spectroscopy at the University of Lille (Laboratoire de Spectrochimie Infrarouge et Raman, Universite des Sciences et Techniques de Lille Flandres Artois) took the lead at that time in the exploration and development of the promise of Raman microspectroscopy and microscopy. Other research laboratories, principally in the United States, followed suit and virtually in parallel pursued these approaches that would, well before 1975, demonstrate the utility of Raman microprobing through the use of the first generation of prototype Raman microprobes. In this exciting development of the technique, the workers at Lille were widely recognized as the pioneers of this emerging field. During this same time, the microanalytical techniques of ion microprobe/microscopy (based on secondary ion mass spectrometry, SIMS) were developed in France, and the development of laser microprobe mass spectrometry (LAMMS) was undertaken in Germany. These latter techniques, initially furnishing only elemental composition information at high sensitivities, also had the advantage of allowing for isotopic discrimination. It is against this backdrop of the scientific scene, over the past 25 years, that this book sets the stage for a thorough discussion of the important aspects of Raman microspectroscopy and microscopy. The book is laid out in ten chapters addressing the fundamental principles of Raman spectroscopy, their application to the concept of Raman microanalysis, the design and construction of micro-Raman instrumentation, and the application of such instruments to a broad spectrum of problems in materials science. Throughout these discussions, the contributing authors highlight the unique aspects of the technique, emphasizing their analytical strengths and limitations, and placing the material in the wider context of modern methodologies for comprehensive materials characterization. From this perspective, the topics presented should fulfill a variety of needs facing both the newcomer to the field as well as the researcher famihar with the analytical uses of vibrational spectroscopy, be that in an academic environment or in an industrial laboratory setting. As with any edited book, the reader will note differences in style as well as an in-depth coverage of specific topics presented. This in no way distracts from the value of the book but rather underscores the different levels of utility that can be assigned to the treatment of this subject. Each chapter emerges as an excellent guide to the sub-topic that is presented. Where rigorous treatment is required, as in the discussion of fundamental principles, the criteria of careful optical design in instrumentation, and the demands on analytical performance, the respective authors come forth with authoritative insight. The book becomes especially useful through the extensive references to the published literature. Foremost, the book is intended for the analytical microspectroscopist using the vibrational spectrum (and this does not exclude the professed infrared spectroscopist) as the diagnostic fingerprint. Yet, microscopists from other disciplines will find this

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work directly pertinent. This can be asserted with assuredness, as the modern research environment increasingly relies on all available probe techniques for multidisciplinary materials characterization. A selective focus may be given on the main thrusts presented in the book. The authors clearly did not intend for any chapter to be an all-encompassing text but rather emphasize the key issues and their consequences of analytical importance. Chapter 1 provides a concise treatment of the normal or spontaneous Raman effect in the context of classical light scattering, the excitation of molecular vibrations, and the appearance of the Raman spectrum. Discussed are polarization effects that require careful attention in Raman microsampUng, a theme that recurs in subsequent chapters on the design of microRaman fore-optics and its effects on the observed spectrum. Discussed also are non-linear Raman effects, specifically resonance Raman scattering, as they often come into play in actual measurements. Laser-excited fluorescence and luminescence are acknowledged as being among the most troublesome spectroscopic interferences encountered by the analyst. Much attention is given to this aspect also in the appHcations sections, and various strategies are outlined to either minimize or circumvent these effects that are a potential detriment to successful Raman microanalysis and imaging. Chapter 2 underscores the principal characteristics of Raman microspectroscopy and microscopy. Discussed are the requirements for the efficient excitation and collection of the Raman radiation with respect to the spatially resolved microscopic sampling volume. These considerations are out of necessity linked to various possible constraints, such as those presented by the optical properties of the sample, with special emphasis on the comphcations from optical absorption. The essential features of confocal microscopy are discussed since these represent an important recent development in the optical design and performance of various forms of microscopy which now have been embodied in Raman microscopy as well. The most advanced Raman microprobes/microscopes will feature the confocal characteristics to permit optical sectioning of the sample through efficient spatial filtering and improved depth-of-field. Chapter 3 represents the tour deforce on the broad subject of micro-Raman instrumentation. It may well comprise the central treatise of this book as it addresses a diversity of aspects central to micro-Raman methodology. Tied together, in authentic rigor and detail, are the critical design and performance characteristics of all major systems and sub-systems that comprise the functional Raman system, be it for the recording of microprobe spectra or the acquisition of digital Raman images. The treatment of the various topics is effectively aided by numerous illustrations, mainly in the form of optical schematics and diagrams. Special coverage is given various interferometric techniques utilizing Fourier-transform methods of spectral analysis which

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have entered the micro-Raman field, through the experiences from FTinfrared spectroscopy, since the late 1980s. Thus, FT-Raman microspectroscopy is discussed as a novel approach to more successfully deal with the minimization or elimination of sample fluorescence, through excitation at wavelengths in the near-infrared. Discussed in this same context are the astonishing advances made in recent years in Raman instrumentation based on the development of dispersive Raman spectrometers and spectrographs with superb stray light rejection, through revolutionary methods of optical filtering, and highly efficient energy throughput. These instruments are coupled to high-sensitivity photoelectric detectors (common types are the IPDA, CCD, or CID) to allow for efficient Raman excitation and detection beyond the traditional visible wavelengths, now extending into the nearinfrared. A most interesting and useful extension of this chapter is the discussion of digital signal processing. The basic mathematical relationships are presented for the sampling and processing of analog spectral data, the Fourier-transform treatment, and the practical aspects of the digitization of Raman spectra. Chapter 4 concerns the topic of Raman imaging. Current imaging methods are classified as either 'parallel' or 'direct imaging' or 'series imaging' techniques. The early attempts at direct imaging Raman methods were based on the same principles of image generation as are used in x-ray and ion-probe microanalysis. Regardless of the specific approach at Raman imaging, the object is to obtain 2D- or 3D-images that provide information on the spatial compositional distribution (for purposes of compositional mapping) of one or more components of the sample. In all cases, imaging capabilities built into a micro-Raman system require relatively sophisticated techniques and these have been adapted by commercial instruments over the past five years. The various technologies employed for Raman imaging are in a great state of flux and presently experience profound changes with the remarkable development of holographic filters, the introduction of acousto-optic tunable filters, and the continued improvement of two-dimensional detectors. Current advances in this range of technologies now permit true confocal image generation and good image contrast formation for at least major constituents of a sample matrix. The topic of Raman microscopy in combination with other microanalysis techniques is discussed in Chapter 5. The French workers have always toyed with the idea of combining two or more microprobe principles and embodying them in the same instrument. These possibihties have been explored and their implementation worked out in considerable detail for the union of Raman microscopy with: (i) electron microscopy/x-ray microanalysis; (ii) ion microprobe mass spectrometry; and (iii) laser microprobe mass spectrometry.

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The conceptualization of these conjugated microanalytical techniques presents no great intellectual challenge. However, their realization and implementation into a practical analytical tool is quite another. ReaUstic design considerations and performance attributes are set forth for each of these dual-use microprobe systems, the aim being not to compromise the performance of either the Raman probe or the second probe function of the coupled instrument. The concept has been fully realized in the construction of a prototype coupled micro-Raman/electron probe instrument. The other two variants of a dual-use microprobe system have so far remained on the drawing board, though there are no outright technical obstacles that would prevent the construction of prototypes to demonstrate feasibiUty. The remaining five chapters of this volume cover the appUcations of Raman microscopy in various fields of chemistry and physics, the geological and environmental sciences, biology and medicine, and closing with a chapter outside the typical realm of either the natural or life sciences. This last chapter gives selected examples from the investigation of art objects, the characterization of gems, and cases from forensic science. Micro-Raman researchers, from the early days, have attempted to explore the full range of analytical applications, in part to define the limitations of the technique. These efforts, from laboratories world-wide, have resulted in an extensive pubUshed Uterature. In their task to review and focus on specific fields of application, the authors had to be selective in their choice of discussion topics and in the extent and depth of coverage. In great measure, this goal has been achieved, so that the broad spectrum of applications fully documents the wide-ranging analytical utility of Raman microscopy. It is not possible here to even provide a narrow, Umited focus on several of the areas chosen. The appUcations delve into formidable research problems from the realm of high-technology materials, such as high-Tc superconductors, to probing the molecular make-up of single living cells. Even the reader who brings only a limited understanding to one or more of these areas of application, will find much useful, and often tantalizing, information in the coverage of these chapters. In closing this overview of the book, some concluding comments may be passed on. The period of development of Raman microscopy represents an interesting time for anyone associated with modern methods of microanalysis applied to materials science. The Lille pioneers of this field were greatly helped, along these avenues of progress, by many other researchers world-wide. In summary, then, as is reflected by this book, the present state of Raman microscopy is the outcome of an exciting and intensive team effort, marked by plenty of cross-fertilization. In view of this, one can conclude that the future of this field will hold no lesser accomplishments and discoveries. I thank the editors of this book, and the contributing authors, for allowing

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me an early look at this work and giving me the opportunity to comment, in this Preface, on the content of this volume. Edgar S. Etz Chemical Science and Technology Laboratory National Institute of Standards and Technology Gaithersburg, Maryland 20899, USA April 1996

Acknowledgements

The Editors wish to thank all of the contributors to this volume. Their efforts have made it possible for us to cover the field of Raman microscopy as widely as we are able. We are honored that Dr Edgar Etz of the National Institute of Standards and Technology, one of the founders of this spectroscopic technique, has accepted to write the Preface. The aid in the production of this work which was provided by many members of the research and technical staff of our laboratory is greatly appreciated. We are especially indebted to Professor Paul Dhamelincourt for his contributions to this book and for his invaluable editorial help. Thanks are also extended to Mme Irene Lepreux, who typed many of the contributions - some from handwritten texts. The publisher and the authors wish to thank the following copyright holders who have kindly granted permission to reprint or adapt the illustrations cited. Chapter 2 Figures 1-5 are reproduced by permission of Springer-Verlag GmbH & Co. KG, from G. Turrell (1989). In: Practical Raman Spectroscopy, D. J. Gardiner and P. R. Graves (eds), chapter 2. Chapter 3 Figures 51-53 are reproduced by permission of DILOR from Technical Documentation (1991). Chapter 4 Figures 1, 2, 7 and 9 are reproduced by permission of John Wiley & Sons, Ltd from Barbillat, J., Dhamelincourt, P., Delhaye, M. and Da Silva, E. (1994). /. Raman Spectrosc. 25, 3. Figure 3 is reproduced by permission of Hiithig & Wepf Verlag, Basel, Switzerland, from Batchelder, D. N., Cheng, C , Miiller, W. and Smith, B. J. E. (1991). Makromolekulare Chemie-Makromolecular Symposia 46, 171.

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Figures 4 and 13 are reproduced by permission of the Society for Applied Spectroscopy from Puppels, G. J., Grond, M. and Greve, J. (1993). Appl Spectrosc. 47, 1256. Figures 5 and 17 are reproduced by permission of the Society for AppHed Spectroscopy from Treado, P. J., Levin, I. W. and Lewis, E. N. (1992). Appl. Spectrosc. 46, 1211. Figure 6 is reproduced by permission of the Society for AppUed Spectroscopy from Battey, D. E., Slater, J. B., Wludyka, R., Owen, H., Pallister, D. M. and Morris, M. D. (1993). Appl. Spectrosc. 47, 1913. Figures 8 and 12 are reproduced by permission of John Wiley & Sons, Ltd from Bowden, M., Gardiner, D. J., Rice, G. and Gerrard, D. L. (1990). /. Raman Spectrosc. 21, 37. Figure 10 is reproduced by permission of Elsevier Science Ltd from Treado, P. J. and Morris, M. D. (1990). Spectrochim. Acta 13, 355. Figure 11 is reproduced by permission of S. Hirzel Verlag GmbH & Co. from Dhamelincourt, P. and Bisson, P. (1977). Microscop. Acta 79, 267. Figure 14 is reproduced by permission of the Society for AppUed Spectroscopy from Batchelder, D. N. and Cheng, C. (1993). Appl. Spectrosc. 47, 922. Figure 16 is reproduced by permission of the Society for AppUed Spectroscopy from Treado, P. J., Govil, A., Morris, M. D., Sternitzke, K. D. and McCreery, R. L. (1990). Appl. Spectrosc. 44, 1270. Chapter 5 Figures 18-22 are reproduced by permission of Springer-Verlag GmbH & Co. KG, from Turrell, G. (1989). In: Practical Raman Spectroscopy, D. J. Gardiner and P. R. Graves (eds), chapter 2. Chapter 6 Figures 9 and 10 are reproduced by permission of the publication board of the Japanese Journal of Applied Physics, from Mizoguchi K. Nakashima S., Fujn, A., Mitsuishi, A., Miromoto, H., Onada, H. and Kato, T. (1987) Jpn J. Appl. Phys. 26, 903. Figure 11 is reproduced by permission of the American Institute of Physics, from Mizoguchi, K., Harima, H., Nakashimi, S. I. (1995). J. Appl. Phys. 11, 3388. Figures 12 and 13 are reproduced by permission of the American Institute of Physics, from Nakashima, S., Inoue, Y. and Mitsuishi, A. (1984). / . Appl. Phys. 56, 2989. Figure 14 is reproduced by permission of the publication board of Oyo-Buturi from Mizoguchi, K., Nakashima, S., Inoue, Y., Miyauchi, M. and Mitsuishi, A. (1986). Oyo-Buturi 55, 73. Figures 18 and 19 are reproduced by permission of the American Institute of Physics from Nakashima, S., Yugami, H., Fujii, A., Hangyo, M. and Yamanaka, H. (1988). /. Appl. Phys. 64, 3067.

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Chapter 7 Figure 1 is reproduced by permission of Springer-Verlag GmbH & Co. KG, from Mernagh, T. P. and Liu, L. G. (1991). Phys. Chem. Minerals 18, 126. Figure 2 is reproduced by permission of Macmillan Magazines, Ltd, from Smith, D. C. (1984). Nature 310, 641. Figure 3 is reproduced by permission of Springer-Verlag GmbH & Co. KG, from McMillan, P. F., Wolf, G. H. and Lambert, P. (1992). Phys. Chem. Minerals 19, 71. Figure 4a,b is reproduced by permission of Springer-Verlag GmbH & Co. KG, from Velde, B., Syono, Y., Kikuchi, M. and Boyer, H. (1989). Phys. Chem. Minerals 16, 436. Figure 4c is reproduced by permission of the American Geophysical Union from Velde, B. and Boyer, H. (1985). /. Geophys. Res. 90, 3675. Figure 5 is reproduced by permission of Pergamon Press, Ltd, from Virag, A., Wopenka, B., Amari, S., Zinner, E., Anders, E. and Lewis, R. L. (1992). Geochim. Cosmochim. Acta 56, 1715. Figure 6 is reproduced by permission of Plenum Publishing Co. from Etz, E. S., Rosasco, G. J. and Blaha, J. J. (1978). In: Environmental Pollutants, T. Y. Toribara and J. R. Coleman (eds), p. 413. Figures 7a and 8 are reproduced by permission of the Americal Geophysical Union from Hemly, R. J. (1987). In: High-pressure Research in Mineral Physics, M. H. Manghnani and Y. Syono (eds), pp. 347 and 355. Figure 7b is reproduced by permission of the Mineralogical Society of America from McMillan, P. and Akaogi, M. (1987). Am. Mineral. 72, 361. Figure 7c is reproduced by permission of Springer-Verlag GmbH & Co. KG, from McMillan, P. and Ross, N. L. (1987). Phys. Chem. Minerals 14, 225. Figure 7d is reproduced by permission of Springer-Verlag GmbH & Co. KG, from McMillan, P., Akaogi, M., Ohtani, E., Wilhams, Q., Nieman, R. and Sato, R. (1989). Phys. Chem. Minerals 16, 428. Figure 7e is reproduced by permission of the Americal Geophysical Union from Hemley, R. J., Cohen, R. E., Yeganeh-Haeri, A., Mao, H. K., Weidner, D. J. and Ito, E. (1989). In: Perovskite: A Structure of Great Interest to Geophysics and Materials Science, A. Navrotsky and D. J. Weidner (eds), p. 35. Figure 9 is reproduced by permission of the American Chemical Society from Sato, R. K. and McMillan, P (1987). /. Phys. Chem. 91, 3494. Figure 10 is reproduced by permission of the American Geophysical Union from Gillet, P., Richet, P., Guyot, F. and Fiquet, G. (1991). /. Geophys. Res. 96, 11 805. Figure 11a is reproduced by permission of the Mineralogical Society of America from McMillan, P. (1985). In: Microscopic to Macroscopic.

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Atomic Environments to Mineral Thermodynamics, S. W. Kieffer and A. Navrotsky (eds), P. H. Ribbe, Reviews in Mineralogy 14, 9. Figure llb,c is reproduced by permission of the American Physical Society from Shapiro, S. M., O'Shea, D. C. and Cummins, H. Z. (1967). Phys. Rev. Lett. 19, 361. Figure 12 is reproduced by permission of Scanning Microscopy International from Beny-Bassez, C. and Rouzaud, J. N. (1985). Scanning Elec. Micros. 1, 119. Figure 13 is reproduced by permission of the American Physical Society from Hemley, R. J. and Mao, H. K. (1988). Phys. Rev. Lett. 61, 857. Figures 14 and 21 are reproduced by permission of E. Schweizerbart'sche Verlagsbuchhandlung from Dubessy, J., Boiron, M. C , Moissette, A., Monnin, C. and Sretenskaya, N. (1992). Eur. J. Mineral. 5, 885. Figure 15 is reproduced by permission of Pergamon Press, Ltd, from Schiffries, C. M. (1990). Geochim. Cosmochim. Acta 55, 721. Figure 16 is produced by permission of the Mineralogical Society (UK) from Guilhaumou, N., Jouaffre, D., Velde, D. and Beny, C. (1990). Bull. Mineral. I l l , 517. Figure 17 is reproduced by permission of Pergamon Press, Ltd, from Pironon, J., Sawatzki, J. and Dubessy, J. (1991). Geochim. Cosmochim. Acta 55, 3885. Figure 18 is reproduced by permission of Elsevier Science Pubhshers from Zhang, Y. G. and Frantz, J. D. (1992). Chem. Geol. 100, 51. Figure 20 is reproduced by permission of San Francisco Press, Inc., from Pasteris, J. D., Seitz, J. C , Wopenka, B. and Chou, I.-M. (1990). In: Microbeam Analysis, R. Geiss (ed.), p. 228. Figure 22 is reproduced by permission of E. Schweizerbart'sche Verlagsbuchhandlung from Dubessy, J., Poty, B. and Ramboz, C. (1989). Eur. J. Mineral. 1, 517. Figure 23a is reproduced by permission of Pergamon Press, Ltd, from McMillan, P., Piriou, B. and Navrotsky, A. (1982). Geochim. Cosmochim. Acta 46, 2021. Figure 23b is reproduced by permission of the American Institute of Physics from Furakawa, T., Fox, K. E. and White, W. B. (1981). /. Chem. Phys. IS, 3226. Figure 23c is reproduced by permission of the Societe Frangaise de Mineralogie et Cristallographie from McMillan, P. and Piriou, B. (1983). Bull. Mineral. 106, 57. Figure 24 is reproduced by permission of the American Physical Society from Hemley, R. J., Mao, H. K., Bell, P. M. and Mysen, B. O. (1986). Phys. Rev. Lett. SI, lAl. Figure 25 is reproduced by permission of the American Institute of Physics from Wolf, G. H., Durben, D. J. and McMillan, P. (1990). /. Chem. Phys. 93, 2280.

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Figure 26 is reproduced by permission of Elsevier Science Publishers from Mysen, B. O. and Frantz, J. D. (1992). Chem. GeoL 96, 321. Chapter 9 Figures l b and 4 are reproduced by permission of Macmillan Press, Ltd, from Puppels, G. J., de Mul, F. F. M., Otto, C , Greve, J., Robert-Nicoud, M., Arndt-Jovin, D. J. and Jovin, T. M. (1990). Nature 347, 301. Figures 2 and 3 are reproduced by permission of Springer-Verlag GmbH & Co. KG, from Puppels, G. J., Olminkhof, J. H. F., Sergers-Nolten, G. M. J., Otto, C., de Mul, F. F. M. and Greve, J. (1991). Exp. Cell Res. 195, 361. Figure 5 is reproduced by permission of the National Academy of Sciences (USA) from Yu, N.-T., Cai, M.-Z., Ho, D. J.-Y. and Kuck, J. F. R., Jr. (1988). Proc. Natl Acad. Sci. USA 85, 103. Figure 6 is reproduced by permission of Academic Press, Inc., from Bot, A. C., Ashkin, A. and Dziedzic, J. M. (1987). Science 235, 1517. Figure 17 is reproduced by permission of the Royal Society of Chemistry from Manfait, M., Morjani, H., Efremov, R., Angiboust, J.-F., Polissiou, M. and Nabiev, I. (1991) In: Spectroscopy of Biological Molecules, R. E. Hester and R. B. Girling (eds), p. 303. Figure 18 is reproduced by permission of the Royal Society of Chemistry from Millot, J.-M., Morjani, H., Aubard, J., Pantigny, J., Nabiev, I. and Manfait, M. (1991). In: Spectroscopy of Biological Molecules, R. E. Hester and R. B. Girling (eds), p. 305. Figures 19 and 20 are reproduced by permission of Springer-Verlag GmbH & Co. KG, from Nabiev, I., Morjani, H. and Manfait, M. (1991). Eur. Biophys. J. 19, 311. Chapter 10 Figures 1, 3, 5, 6, 7 and 9 are reproduced by permission of Palais de la Decouverte (Paris) from Coupry, C. (1992). Revue du Palais de la Decouverte 20(196), 15. Figure 2 is reproduced by permission of Centre d'Etude des Manuscrits, Bibliotheque Royale (Bruxelles) from Guineau, B., Coupry, C , Gousset, M. T., Forgerit, J. P. and Vezin, J. (1986). Scriptorium XL, 157. Figure 8 is reproduced by permission of John Wiley & Sons from Coupry, C , Lautie, A., Revault, M. and Dufilho, J. (1994). / . Raman Spectrosc. 25, 92. Figure 10 is reproduced by permission of Association frangaise de Gemmologie from Dele-Dubois, M. L., Poirot, J. P. and Schubnel, H. J. (1986). Rev. Gemmologie 88, 15. Figure 11 is reproduced from Dubois-Fournier, M. L. (1989). Diplome de Gemmologie, Universite of Nantes, France. Figure 12 is reproduced by permission of Elsevier Science Publishers from

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Dele-Dubois, M. L., Dhamelincourt, P., Poirot, J. P. and Schubnel, H. J. (1986). /. Mol Struct. 143, 135. Figure 13 is reproduced by permission of Association frangaise de Gemmologie from Dele-Dubois, M. L., Dhamelincourt, P. and Schubnel, H. J. (1981). Rev. Gemmologie 63, 11.

1 The Raman Effect George Turrell

I. INTRODUCTION

The subject of this book is the Raman effect, a phenomenon which results from the interaction of hght and matter. In particular, the book is concerned with the small world of microcrystals, microorganisms, microelectronics, etc., some of those areas of application in which Raman microspectroscopy enjoys wide acclaim. This introduction to the various apphcations and methods of this technique therefore includes an analysis of the interaction of electromagnetic radiation with molecular systems. The scattering of light, as a result of its interaction with matter, can be classified as elastic (Rayleigh or Mie-Tyndall scattering) or inelastic (Raman or Brillouin scattering). In the former case the scattered light is observed at the same frequency as the incident Ught. On the other hand, inelastically scattered light, which is detected at different frequencies, constitutes the Raman or Brillouin spectrum of the sample.

II. HISTORY OF THE RAMAN EFFECT

Before addressing the scientific problem at hand, it would seem appropriate to recall its historical background. The inelastic scattering of hght by matter was predicted on theoretical grounds by Brillouin (1922) and by Smekal (1923). It was included in the Kramers-Heisenberg theory of second-order optical phenomena (1925). The first experimental observation of the inelastic scattering of light was made by Raman and Krishnan (1928). The experimental setup consisted of a source - a focused, filtered beam of sunUght; a sample - a large volume of a neat Hquid; and a detector - the human eye! The basic experimental arrangement has not varied significantly since that time.

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G. Turrell

The first qualitative observations were very rapidly confirmed and placed on a quantitative basis by Cabanes (1928), Landsberg and Mandelstram (1928), Rocard (1928) and by Raman and Krishnan (1929). A complete semi-classical theory of the Raman effect was published a few years later (Placzek, 1934). In spite of the initial excitement over its discovery, in the period between the experiments of Raman and Krishnan and the first use of laser excitation (Porto and Wood, 1962; Stoicheff, 1963), the spectroscopic application of the Raman effect made relatively little progress. Its role was limited to a somewhat esoteric complement to infrared spectroscopy. The renaissance of Raman spectroscopy was inspired by the invention of the laser (Schawlow and Townes, 1958; Maiman, 1960), the ideal source for Raman spectroscopy; through the following years, a considerable number of instrumental developments were made. Among them should be mentioned the fabrication of high-quality holographic gratings, improved detectors including multielement arrays - and efficient computer treatment of experimental data. More recently, the application of Fourier transform methods to Raman spectroscopy has shown considerable promise, particularly in the suppression of interference due to sample fluorescence. The birth of Raman microspectroscopy dates from 1966, when Delhaye and Migeon (1966) published two often overlooked papers in which they pointed out that the intensity of Raman scattered light should not decrease with decreasing sample volume, as might be intuitively expected. In fact, these authors showed that the intensity remains constant with decreasing sample size, down to dimensions determined by the diffraction Hmit, and hence the wavelength of the laser excitation. Within a few years the basic principles of Raman microspectroscopic instrumentation were defined (Hirschfeld, 1973) and, soon after, two different Raman microspectrometer systems were described (Delhaye and DhameUncourt, 1974; Rosasco et al., 1974). The former instrument, which was subsequently commercialized, provides for Raman imaging (mapping), as well as single-point analysis.

III. MECHANISM OF THE RAMAN EFFECT

The Raman effect results from the interaction of vibrational and/or rotational motions of molecules with the electromagnetic radiation, while Brillouin scattering involves the translational motion of molecules in liquids and solids. The latter effect, which produces only very small frequency shifts, and which has not as yet yielded important appHcations, will not be considered in this volume. A simple classical picture of the Raman effect can be obtained by analogy with the ampHtude modulation of a radiofrequency carrier wave by an audio

The Raman Effect

3

signal. The resulting sidebands are similar to the Raman spectrum produced by the combination of the frequencies of molecular vibrations with the frequency of the laser excitation. However, for most purposes a quantum mechanical model is more useful. According to quantum theory, a molecular motion can have only certain discrete energy states. A change in state is thus accompanied by the gain or loss of one or more quanta of energy. A quantum of energy is defined by A£ = hv}^, where h in Planck's constant and v^ is the classical frequency of the molecular motion. The interaction of a molecule with electromagnetic radiation can thus be analyzed in terms of an energy-transfer mechanism. For example, the simplest absorption process involves the gain of a quantum of energy by the molecule, accompanied by the annihilation of a quantum of light or photon. Similarly, spontaneous emission can be described as the creation of one or more photons due to the corresponding loss in molecular energy. Scattering processes involve at least two quanta acting simultaneously in the light-matter system. Simple elastic scattering occurs when a quantum of electromagnetic energy is created at the same time that an identical one is annihilated. Thus, the molecule is unchanged by the event. In the case of an inelastic process such as the Raman effect, the two photons are not identical and there is a net change in the state of the molecule. If, for example, the created photon is less energetic than the annihilated one, the scattered light is observed at a frequency that is lower than that of the incident light. This case is referred to as Stokes Raman scattering. On the other hand, if the created photon is the more energetic of the two, the Raman frequency will be higher than that of the laser and the anti-Stokes spectrum will be produced. The scattering processes described above are illustrated in Fig. 1. The laser excitation at frequency VQ reappears as the relatively strong Rayleigh line. The much weaker Raman 'sidebands' are the result of inelastic scattering by, say, a molecular vibration of frequency v^. It should be emphasized that the efficiencies of these scattering processes are very low. Typically, the intensity of the Rayleigh line is about 10""^ with respect to the incident excitation, while the Raman features are at least another factor of 10"^ weaker. It should be obvious from Fig. 1 that the Raman frequencies can be measured relative to that of the excitation. Thus, the origin of the abscissa scale in Fig. 1 can just as well be placed at the position of the excitation frequency and the Raman frequencies will then appear at ±Vy. In practice, as a vibrational frequency has a value of the order of 10^^ s~^, the frequency values are usually divided by the velocity of fight expressed in cms~^. The resulting quantity is then a wavenumber in units of cm~^, which is defined by P v = Vy/C=

1/Av,

where Ay is the corresponding wavelength.

(1)

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G. Turrell

> 0)

z

RAYLEIGH

iU

o

f^ FREQUENCY,V

Figure 1 Raman and Rayleigh scattering of excitation at a frequency VQ. A molecular vibration in the sample is of frequency v^.

Again referring to Fig. 1, a single Raman band is shown on each side of the Rayleigh line. This spectrum is representative of the spectrum of a diatomic molecule, which has but one vibrational frequency, in the liquid state, where rotational motion of the molecule is usually suppressed by the intermolecular forces. The spectrum in more conventional form is shown in Fig. 2a. Note that the direction of the abscissa scale has been reversed so that the Stokes spectrum appears on the right-hand (positive) side. Diatomic molecules in the gas phase at moderate pressures rotate freely and the resulting modulation of the Raman bands due to this quantized motion is represented in Fig. 2b. Note here the 'pure rotational' structures on each side of the Rayleigh line, as well as the rotational branches on each side of the vibrational Raman bands. In the solid state the rotational motion of piolecules is usually restricted to oscillatory-type 'librational' modes which

The Raman Effect

5

RAYLEIGH (a) STOKES ANTI-STOKES

A

5^:=^ vo

Vv

-Vv

RAYLEIGH (b) STOKES

ANTI-STOKES

JM Vv

m.

•//AAI>LW

-Vv

Vo WAVENUMBER SHIFT

Av (cm~^)

Figure 2 Raman and Rayleigh spectra typical of a diatomic molecule, (a) In the liquid phase, (b) In a gas, where rotational structure becomes apparent. Note that the abscissa scale is expressed in wavenumbers with respect to the excitation frequency. may appear as weak satellite feaatures. Furthermore, in crystals, splitting of vibrational bands may occur, depending on the structure of the unit cell (Turrell, 1972).

IV. ELECTROMAGNETIC RADIATION AND CLASSICAL LIGHT SCATTERING The propagation of electromagnetic radiation is described by Maxwell's equations, which determine the behavior of the electric and magnetic fields in space and time. For the simple case of a plane-polarized wave in a homogeneous medium these fields have, at a particular instant in time, the

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G. Turrell

%^!H

Figure 3 A plane-polarized electromagnetic wave in a homogeneous medium.

forms shown in Fig. 3. Here, %, the electric vector, is directed along the X axis and the magnetic vector !H is parallel to the Y axis. The direction of propagation of the wave is specified by the Poynting vector S = %X!H, and is thus in the Z direction. The electric field, which is of primary importance in the analysis of scattering phenomena, can be represented in this case by "^x^ '^A'exp(-a>o/cZ/c) exp[-\a)Q{nZ/c - t)],

(2)

where OJQ = ITTVQ and VQ is the frequency of the light. The real and imaginary parts of the index of refraction of the medium are given by n and K, respectively; c is the velocity of light in free space and t is the time. This radiation is said to be plane polarized in that the electric field is always in the same direction {X). The amplitude of the wave is %^ and the origin of the Z axis is arbitrary. The factor exp[-ict)o(AzZ/c -1)] in Eq. (2) is periodic in both space (Z) and time, and the velocity of propagation of the wave is given by c/n. The factor exp[-a)o KZ/C)] expresses the loss in electromagnetic energy due to absorption by the medium. Thus, the absorption coefficient or absorptivity which enters into a Beer's law analysis of absorption

The Raman Effect

7

pheomena is proportional to K, the imaginary part of the refractive index of the medium. In free space (a lossless medium) n = \ and /c = 0. The effect of the electric field on a molecule is to polarize the electron distribution. Thus, a dipole moment is induced in the molecule. If the electric field is not too strong, the induced moment is given by y^ = ^%,

(3)

where a is the polarizability of the molecule. Under more intense radiation, terms in %^, %^, etc. must be added in order to account for hyper-Raman effects, which will not be considered here. As both |JL and % in Eq. (3) are vector quantities, the polarizability is a tensor. If magnetic phenomena are not involved, it is composed of nine real elements. The form of the tensor depends on the coordinate system chosen and the molecular symmetry. Because both |x and % are time-dependent, the induced dipole moment oscillates in time, leading to emission of radiation - the classical model of the scattering processes. A simpHfied illustration of this model is instructive. The time dependence of ^j^can be represented by, %x= %%:CO%i27Tvt), where the real part of Eq. (2) has been taken. A diatomic molecule vibrates at a frequency v^ and, assuming simple harmonic motion, its internuclear distance can be written in the form q^ = q^ cos {lirv^t), where q^ is the amplitude of vibration. The polarizability, which in this case is simply a scalar quantity, can be expanded as a Taylor series in q^. Then, n

/ da ,

Mvjo oL^+[^\

(4)

qycosilrrv^i),

where higher terms are neglected for small atomic displacements. Substitution of Eq. (4) into Eq. (3) leads to JJL = %%a^ c o s ilTTV^t) + %%{ - ^ 1

(7^ COS ( 2 W Q O COS (2771^^0

(5)

Va<7v/o = %^xOL^cos{27TVoi) + %^ ~ \ \d^v/o

^^{cos[(27r(^o" ^v>] + cos[(277(^o+ ^v)0(6)

The first term on the right-hand side of Eq. (6) represents the oscillation of the induced dipole at the frequency VQ of the incident light, resulting in Rayleigh scattering. The vibrational sidebands referred to above (Raman scattering) are produced at frequencies VQ — Vy (Stokes) and VQ -h Vy (antiStokes), as shown in Fig. 1. It is important to note that the second term in Eq. (4) contains the factor (da/d^v)o. The intensity of the Raman features

8

G. Turrell

are thus dependent on the derivative of the polarizabihty with respect to the molecular coordinate q^^. This principle will be developed later in this chapter. The above picture of electromagnetic radiation is a classical one. The analysis of its interaction with a quantized molecular system leads to the usual semi-classical description of the absorption of light. However, a more sophisticated model of electromagnetic radiation is based on quantum electrodynamics. In the latter theory the electromagnetic field is also quantized. The treatment of its interaction with molecular systems is considerably simpler than the semi-classical one. In particular, the equations which describe both spontaneous and induced emission evolve automatically. Furthermore, the various two-photon processes of interest here are much easier to handle within the framework of this model. Unfortunately, the quantum field theory is not generally well known among chemists, although its advantages, particularly in the analysis of resonance Raman phenomena, is unquestionable (Hameka, 1965).

V. MOLECULAR VIBRATIONS The diatomic molecule considered in Fig. 1, as an illustration of the classical Raman effect, was characterized by a vibrational frequency v^. However, the vibrational energy is, of course, quantized in the sense that the vibrational quantum of energy is equal to A£ = hvy^. For a polyatomic molecule consisting of N atoms, the problem is analogous, although it is somewhat more complicated. The presence of A^ atoms results in 3A^ degrees of freedom, the number of coordinates necessary to specify the positions of all N atoms. However, for a molecule in free space, as in a perfect gas, three coordinates are used to define the position of the center of gravity of the molecule. These three coordinates are sufficient to describe the translational motion of the molecule. Furthermore, for a nonlinear molecule, three additional coordinates (for example, Euler's angles) are necessary to specify the orientation of the molecule in space. (In the case of a linear molecule two orientational coordinates are sufficient.) The result of these arguments is that 3N(total) - 3(translational) - 3(orientational) = 3A^ — 6 vibrational degrees of freedom are available for each nonlinear molecule. (For a linear molecule the corresponding result is

3N-5.) These 3A^ — 6 vibrations of a nonlinear polyatomic molecule can be described classically by a corresponding number of normal modes. The advantages of this description are, first, that each normal mode has a specific

The Raman Effect

9

vibrational frequency, and second, that in the harmonic approximation the 3N-6 modes are independent. Finally, according to the quantum mechanical picture, each normal mode K leads to a series of vibrational energy levels separated by a quantum of vibrational energy defined by A£K = ^^KWithout any consideration whatsoever of the dynamics of a molecule, it is possible to classify its normal vibrations according to their symmetry. The analysis is based entirely on the geometry of the molecule in its equilibrium configuration, which is characterized by certain symmetry operations. These geometrical operations, or, more correctly, coordinate transformations, leave the molecular energy invariant. Furthermore, the ensemble of symmetry operations forms a group in the mathematical sense. This application of the theory of groups allows the normal modes to be classified according to certain symmetry species (in group theory, irreducible representations) for each group. This subject, which has been treated by a number of authors (see, for example, Wilson et al., 1955), will not be developed here. However, two results of the symmetry classification of molecular vibrations should be mentioned, as they are especially important in the present application. First, there is always one symmetry species in a group which retains all of the symmetry of the equilibrium configuration of the molecule. The vibrational modes which belong to this species are referred to as totally symmetric. All other modes of a given molecule are antisymmetric, or at least asymmetric, with respect to one or more symmetry operations of the group. A second symmetry consideration which is useful in the determination of vibrational selection rules is the inversion. That is, the exchange of all equivalent atoms (or coordinates) with respect to a center of molecular symmetry. In a molecule which possesses a center of inversion, all normal modes of vibration are either symmetric or antisymmetric, g or u (German: gerade, ungerade) with respect to the inversion operation. It is easily shown that in such cases those vibrational modes which are symmetric under inversion {g) are not infrared-active. On the other hand, those which are antisymmetric {u) are inactive in the Raman spectrum. This general principle is referred to as the rule of mutual exclusion. In general, the classification of normal modes of vibration in symmetry species leads not only to certain, albeit limited, descriptions of the forms of the molecular vibrations, but, more importantly, to direct determinations of the selection rules which govern infrared and Raman spectral activity. The normal-mode frequencies depend on the atomic masses, the molecular geometry and the interatomic forces in the molecule. These last quantities are described in the harmonic approximation by the ensemble of force constants which constitute the force field. In most cases the atomic masses and the molecular geometry are known, the latter as a result of electron or X-ray diffraction studies. The force field, on the other hand, is usually not known, although considerable progress has been made in recent years in the

10

G. TurretI

calculation of force constants for small molecules with the use of ab initio or semi-empirical methods. In practice, force constants are determined by the resolution of the secular equations which relate the observed vibrational frequencies of a molecule to its geometry and its intramolecular forces. This calculation will not be discussed here, as the reader will find its description in numerous references (for example, Wilson et al., 1955). However, the force field calculations which have been carried out on many small and medium-sized molecules (some tens of atoms) have yielded reliable values for the constants which are characteristic of many chemical bonds and a number of functional groups. It has been adequately demonstrated that these quantities can, with care, be transferred to similar bonds and groups in other, often much larger, molecules. The result is that fairly realistic dynamical pictures of the behavior of large molecules, in particular those of biological interest, are now being calculated. An additional contribution of the results of force field calculations to the interpretation of vibrational spectra is in the quantitative descriptions of so-called group frequencies. Those frequencies which correspond to normal modes of vibration which are more or less localized in a particular chemically significant group constitute the characteristic frequencies of the group. For example, organic chemists have for years related a strong infrared absorption band in the 1700 cm~^ region to the presence of a carbonyl group in the molecule. Normal-coordinate calculations which have been made on many small carbonyl compounds show that this mode is not completely localized in the C = 0 bond. Thus, the frequency displays its well-known sensitivity to substitution on nearby atoms in the molecule (Bellamy, 1975). The group-frequency concept is essential to the interpretation of the vibrational spectra of very large molecules - in particular, those of biological interest. For example, all amides, polypeptides and proteins exhibit strong features at approximately 1660 cm~^ in the Raman, as well as the infrared, spectra (Dollish et al., 191 A). It was shown a number of years ago by Miyazawa et al. (1958) that N-methylacetamide has a normal vibration at 1660 cm~^ associated with the mode represented in Fig. 4. Although this mode involves principally (80%) the stretching of the C = 0 bond, the C—N stretching and N—H bending coordinates contribute approximately 10% each. This analysis provides the model for the amide I band, which is characteristic of the peptide Hnkage. Variations in the position of this band have been used to determine the geometrical configuration of the molecule being studied. The peptide linkage can also be identified by the presence of the amide 11 and amide III bands. These modes are largely localized in the N—H bending and C—N stretching coordinates, respectively, although these descriptions are approximate. Thus, for example, all three of the 'amide bands' are sensitive to hydrogen bonding. In some cases, the relative intensity of a Raman band, as well as its

The Raman Effect

11

Figure 4 Model of the amide I vibration: the normal mode of A^-methylacetamide at 1660 cm~^ (Miyazawa et al., 1958).

frequency, can provide structural information. The classical example leading to Eq. (6) showed that the intensity of a Raman band due to a molecular vibration depends on the quantity (da/d^v)o. For polyatomic molecules the normal-mode picture suggests that the derivatives (^«i,j/^GK)o determine the intensity of the fundamental band arising from the normal mode Q^. It will be seen below that the intensities are in general proportional to the squares of these derivatives. For small molecules a general principle evolves from consideration of the symmetries of the normal modes, namely, that fundamental Raman bands due to totally symmetric vibrations are usually stronger than the others. For large molecules a group-intensity argument can be developed which is analogous to that outlined above for the vibrational frequencies (Carey, 1982). Thus, for Raman fundamentals which arise from localized normal vibrations, some general principles apply to relative band intensities, namely: (i) A Raman band is more intense for a mode which involves primarily a bond stretching coordinate than for that which is predominantly an angular deformation. (ii) The intensity of a band due primarily to the stretching of a covalent bond increases with bond order. Thus, for example, the bond C = C provides a greater contribution to a band intensity than does the C—C bond. (iii) For a mode which is largely localized in a given bond, the intensity increases with the atomic numbers of the bonded atoms. (iv) If two bond-stretching coordinates are involved in a normal vibration, the Raman fundamental is more intense when the bonds stretch in phase than when their stretching motions are out of phase. Furthermore, in cyclic molecules those modes which are pseudosymmetric - largely 'breathing' - yield relatively strong bands.

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G. TurretI

It must be emphasized here that, in spite of its utiUty, the group-frequency, group-intensity approach involves serious approximations. Accordingly, band assignments based on these arguments must be made and accepted with certain reservations.

VI. THE SCATTERING TENSOR In the traditional Raman experiment the scattered light is observed along a direction perpendicular to that of the excitation. However, in all existing Raman microspectrometer systems the backscattering geometry is employed. Thus, as shown in Fig. 5, the laser beam is directed along, say, the Z axis, and the light scattered in the — Z direction is collected. In practice the incident light is not in the form of a parallel beam, but is focused on the sample by means of a microscope objective. Furthermore, the scattered Hght is collected in a cone whose solid angle is far from zero. These basic optical problems will be considered in detail in the following chapter. For the present, it is useful to consider the ideaUzed case represented in Fig. 5, where both excitation and scattering are assumed to be in the form of parallel beams of light. The direction of polarization of the electric vector -Z %. %

SAMPLE

Figure 5 The backscattering geometry usually used in Raman microspectroscopy.

The Raman Effect

13

must be specified for each beam. Here, the excitation has been assumed to be polarized in the Y direction and the polarization of the scattered Kght can be chosen in either the X or the Y direction with the use of an analyzer. According to Placzek's semi-classical dispersion theory (Placzek, 1934), which is quite adequate for the description of the normal Raman effect, the intensity of the scattered light is given by 3s = /ei^|ee«espda,

(7)

where K = AiP'a^D'^, a^ 11131 and ^ is the wavenumber of the scattered light. The scattered energy per unit time (intensity) into a solid angle dfl is given by 3s while 4 is the energy per unit area per unit time (irradiance) of the excitation incident on the sample. The unit vectors Cg and Cg define the directions of the electric fields of the exciting and scattered radiation, respectively, and a is the scattering tensor. The tensor a is composed of elements of the type {ocij)xY = {nt I ioixY)o I rij),

(8)

where rij represents the ensemble of rotational and vibrational quantum numbers of the initial state and rij those of the final state involved in the transition. The quantity (axY)oy which is the XY component of the electronic polarizability of the molecule in the ground electronic state, is given by

^^ ^ ^ r 1

^r + ^0

^r-^0

J'

where v^ is the average frequency separation between the ground electric state and a given excited vibronic state r. The sum is over all excited states of the system. The elements such as (aij)xY can be arranged in the form of a 3 x 3 tensor analogous to the classical polarizabihty tensor introduced in Eq. (3). The derivation of Eq. (9) is very long and involves a number of approximations which Hmit its validity. In particular, (i) the excitation frequency VQ must be much higher than those associated with any vibrational or rotational transition of the system, and (ii) the excitation frequency must be lower than the frequency of any electronic transition. Under these conditions, and neglecting the possibility of magnetic effects, the scattering tensor is real, symmetric and frequency-independent. As it is then a function only of the nuclear coordinates, it can, in effect, be replaced by the classical polarizabihty tensor employed above, which in a polyatomic

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G. TurretI

molecule is a function of the normal coordinates. It should be noted that the symmetry properties of the polarizability and the scattering tensors are identical, a consideration which is important in the determination of the selection rules for Raman activity. In the following section the analysis applies to a given Raman vibrational fundamental vibration and the matrix of polarizability derivatives will be represented simply by a . The application of the above arguments to the backscattering configuration (Fig. 5) shows that the Raman intensity is proportional to

CpaCe

(0

1

0)

ayx \0LZX

OLYY OLYZ GLZY

1

=

laYx-^ayyl

(10)

OLzzlx^J

where only the two elements ayx and ayy can contribute to Ught scattering in the - Z direction. With the aid of an analyzer to select the direction of polarization of the scattered hght, a particular element of a can be chosen. From Eq. (10) it is clear that if the analyzer is set in the Y direction, the intensity of the scattered light will depend only on the value of ayy. Similarly, an analyzer setting in the X direction will yield a scattered intensity which depends only on ayx. These two cases correspond to measurements of the scattered intensities ^ or 3^, in which the electric vector of the scattered light is respectively parallel or perpendicular to the polarization direction of the excitation. In the above analysis, the laboratory coordinates X, Y,Z were employed. However, it is often more convenient to specify the polarizability tensor with respect to a system of axes x,y,z attached to a molecule in the sample. In the case of single crystals the crystallographic axes usually form a more useful basis. The transformation of the polarizability tensor from one coordinate system to another is made with the aid of the matrix relation. <^xyz = ^ a XYZ

4>.

(11)

Here, the matrix 4> is orthogonal, with elements which are direction cosines expressed in terms of Euler's angles (Wilson et al., 1955). A geometrical interpretation of the polarizability tensor is often employed because of the analogy between a and the moment-of-inertia eUipsoid in classical mechanics. The principal axes of the ellipsoid are determined by the symmetry of the body considered (here, a molecule or a crystal). An axis of three-fold or higher symmetry is necessarily a principal axis of the body. When principal axes are employed, ocxyz is reduced to diagonal form. Furthermore, two special cases can be defined. If two of the diagonal elements are equal, e.g. a^^ = ayy ^ a^^, the ellipsoid is a surface of revolution about the z axis. When a^^^ = ayy = a^^, the ellipsoid degenerates into a sphere and the polarizability is characterized by a scalar quantity.

The Raman Effect 15 VII. POLARIZATION IN GASES AND LIQUIDS It usually can be assumed that the molecules in a gas or a Hquid are randomly oriented. Then Eq. (11) must be averaged over Euler's angles and the individual elements of oL^y^ cannot be evaluated experimentally. However, certain combinations of them which are invariant under coordinate transformations can be determined. The tensor invariants are defined (Sonnich Mortensen and Massing, 1980) in the form 2^ = i i a , , + a^^ + a , , p ,

(12)

and 2 ^ = \{Wxy

+ OLy^? + I OLyz+ OL^y? + k z x + «JCzP}

+ III OLxx - OCyy\^ + I «yy - OLzz? ^ W z z '

«xxP}-

(14)

The quantities 2 ^ (/: = 0,1,2), which are here defined, are proportional to the invariants originally employed by Placzek and are related to other often used parameters (Long, 1977) by X^ = 3o?, t^ = W, and 2^ = iy^. The quantity 2^ represents the isotropic part of the tensor, while the symmetric part of the anisotropy is given by 2^. These invariants enter in the intensity formulae for ordinary Raman spectra. The antisymmetric part of the tensor, which defines 2^, is important in both resonance Raman and electronic Raman spectroscopies. In order to obtain relations between the observed relative intensities of Raman bands and the tensor invariants, it is necessary to average Eq. (11) over the molecular orientation. This calculation can be carried out directly (Wilson et al., 1955), or with the aid of the Clebsch-Gordon coefficients (Sonnich Mortensen and Massing, 1980). The results for the scattering intensities become 3|| = A:(ayy)2=A:(^20 + ^22)

(15)

^^^KiaYx? = Kill'+W^^).

(16)

and

In ordinary (nonresonant) Raman scattering 2^ = 0 and a measurement of 3j^ yields a relative value of the anisotropy. Thus,

where ^aniso

1n ^

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G. Turrell

and

where

aiso-fs".

(18.1)

The quantities 3iso and 3aniso ^re often estimated directly from spectra recorded under the two different polarization conditions, but their intensities should, correctly, be determined by integration over the respective band profiles. The depolarization ratio, which is defined by P = 3±/3||,

(19)

can be written in terms of the tensor invariants in the form, ^=10X« + 4S2

^^^^

for the case in which the molecules are randomly oriented in the sample. In the ordinary Raman effect 2^ = 0 and 0 < p < 3 / 4 for linearly polarized excitation. Furthermore, for Raman bands arising from non-totally symmetric molecular vibrations, 2^ = 0, yielding p = 3/4 for these so-called depolarized bands.

VIII. POLARIZATION IN CRYSTALS The electric field at a point r in a transparent crystal due to a plane, monochromatic wave propagating in a direction KQ is given by « = «Oexp[-27ri(KoT-i/oO]-

(21)

This expression is the three-dimensional generalization of Eq. (2), with K = 0. As this exciting radiation is usually in the visible region of the spectrum, its frequency is very high compared with the frequencies of any of the crystal vibrations. The magnitude of the wave vector KQ is given by ^0 = nvg/c,

(22)

where n is real refractive index and c/n is the velocity of propagation of the wave. The classical treatment of the Raman effect in crystals now follows the development given above for isolated molecules. The polarizability is expanded in the normal coordinates QK= Q%cxp[-27n(KK'r-VKt)l

(23)

The Raman Effect

17

where K^ is the wave vector of lattice wave K. The induced dipole moment is given to first order by, fjL = a 0 ^ ^ e x p [ - 2 7 r i ( K o - r - i^oO] + %''^i^'KQKtx^{-27n[{K^^KK)'V-{v^^VK)t\}.

(24)

K

where a'jc= {dotldQj^Q, as before. This result shows that in a crystal the scattered light is of frequency VQ + vjc and that it propagates in the direction given by KQ + K^^. The Raman effect is produced when v^ is an optical frequency of the crystal and, again, the frequencies are referred to as Stokes and anti-Stokes, depending on the choice of sign. From the above analysis, it is evident that Raman scattering in crystals is governed not only by the conservation of energy, but also by the conservation of momentum. The latter condition requires that Ko = Ks + K^.

(25)

Thus, as the lattice waves have directional properties, the nature of the Raman spectrum depends on the orientation of the crystallographic axes with respect to the direction and polarization of both the excitation and the scattered Hght. The orientation of a single crystal in a given Raman experiment is generally specified by the Porto notation (Damen et al., 1960). Three directions x, y and z in the crystal are defined which are not necessarily coincident with the crystallographic axes. If the crystal is oriented so that the axes x,y,z are collinear with the space-fixed axes X, Y, Z, the two configurations described by Fig. 5 are represented by z(yy)'z and z(yx)'z. In the Porto notation, the first symbol defines the direction of propagation of the excitation, while the last one refers to the observation direction. The symbols in parentheses specify, in order, the polarization directions of the exciting and scattered light. Furthermore, they identify the particular element of the Raman tensor which is responsible for the observed scattering. It should be apparent from the above discussion that Raman spectroscopic measurements on single crystals can yield considerably more information than can be obtained from similar studies of liquids or gases. In the latter cases only the two experimental optical configurations are possible, leading to the determination of the depolarization ratio. However, Raman spectroscopic investigations of single crystals can, by suitable choices of orientation, yield relative measurements of the squares of all of the elements of the scattering tensor. A table has been derived (Loudon, 1964, 1965) which presents the form of the scattering tensor for each of the 32 crystal classes (symmetry point groups). This table is particularly useful in the interpretation of the Raman spectra of crystalline samples.

18

G. Turrell

It is usually stated that polarization measurements cannot be made on polycrystalline samples because of reflections, which tend to scramble the electric vectors of both the excitation and the scattered hght. However, in Raman microspectroscopy, it is often possible to select optically a single microcrystal from a polycrystaUine powder and make a polarization measurement. As the crystal orientation is not usually known in this case, the experiment is repeated a number of times on different optically chosen microcrystals until consistent values of the depolarization ratios are obtained. Clearly this method is only applicable if the sample contains microcrystals of dimensions somewhat greater than the diffraction limit of the laser beam - typically of the order of 1 |xm. It should be pointed out here that in the case in which the Raman-active fundamental being investigated is also infrared-active, further considerations enter. Then the dipole derivative {djxIdQi^Q is nonzero and the electric field of the excitation interacts directly with the crystal vibrations. The transverse and longitudinal components of the vibrations have different frequencies, v]^ and 1^^ respectively, whose frequency separation is proportional to {d^ldQj^^ and, hence, to the absorption intensity of the corresponding infrared band. In molecular crystals this effect is usually small. However, in ionic crystals, which often exhibit strong infrared absorptions, the difference V]C—V^K^ which corresponds approximately to the width of the Restrahlen region, can become quite large. In this case, the requirement of simultaneous infrared and Raman activity hmits the phenomenon to piezoelectric crystals. Otherwise, for the ten centrosymmetric crystal classes the rule of mutual exclusion appHes and no fundamental vibration can be both infrared- and Raman-active. In piezoelectric crystals, then, each Raman-active fundamental K predicted by factor-group selection rules leads to a pair of Raman bands at frequencies v]^ and i^^, while only the band at v]^ appears in infrared absorption. As the analysis of Raman polarization in piezoelectric crystals is quite compHcated, the reader is again referred to the review article by Loudon (1964, 1965).

IX. RAMAN BANDSHAPES Thus far in this chapter Raman spectral features have been described in terms of frequency shifts, or, simply, wave numbers relative to that of the excitation. The shape or profile of a Raman band was not considered, although it was pointed out in Section VII that the relative intensity of a Raman band is correctly obtained by integration over the entire spectral profile. It should perhaps be mentioned that absolute intensities of Raman bands are extremely difficult to measure. Thus, one is usually content to

The Raman Effect

19

obtain relative values. These relative intensities can be expressed in terms of electro-optical parameters, which are approximately transferable in a manner analogous to the force constants (Gussoni, 1980). At relatively low pressures the Raman scattering of gases is composed of pure rotational spectra and vibration rotation spectra in the form of 'lines'. The expression 'spectral line' dates from the era of photographic recording of Raman spectra. The resulting lines are the images of the slit employed in the monochromator. As the pressure of a gaseous sample increases, the lines broaden, becoming wider than the spectral shtwidth, and blend together, resulting in the bands which are characteristic of high-pressure gases and liquids. The theory of pressure broadening of spectral lines was developed in particular by Anderson (1949) and, more recently, by Gordon (1966). However, it will not be considered here, as the techniques of Raman microspectroscopy are only rarely applicable to gaseous samples (see, however. Chapters 7 and 10). The theory of Raman bandshapes of liquids has also been developed by Gordon (1966). In the simplest case it apphes to the vibrational spectrum of a linear or symmetric-top solute molecule in a dilute solution. As indicated in Section VII, the Raman spectrum can be separated into isotropic and anisotropic components whose intensities are proportional to 2^ and S^, respectively. In dilute solutions, then, the profile of the anisotropic or depolarized Raman band of the solute molecule yields direct information concerning the temporal evolution of its orientation. The decomposition of the profile into band moments allows the short-time behavior of the molecule to be determined and, in principle, provides an experimental method of measuring solute-solvent interactions. On the other hand, the analysis of the shape of the polarized component of the band yields a value of the vibrational relaxation time of the observed fundamental vibration of the solute molecule. For an authoritative review of the theory of spectral bandshapes in Hquids, the reader is referred to the book by Rothschild (1984). In crystalhne solids the Raman bands are relatively sharp, the widths being due primarily to the anharmonicity of the molecular vibrations. Thus, the bandwidths increase approximately linearly with increasing temperature (Loudon, 1964). However, the presence of impurities, vacancies or other imperfections in the crystal lattice results in additional broadening of the Raman bands. In particular, the vibrational Raman spectra of amorphous materials are characterized by very broad features which have quite generally resisted attempts at quantitative interpretation. An article by Brawer (1975), for example, outhnes this problem. If a general theory of the Raman spectra of amorphous soHds can be developed, the observed band profiles should yield a considerable amount of information concerning the nature and range of disorder, as well as the density of vibrational states, in these materials.

20

G. Turrell

X. RESONANCE RAMAN SCATTERING AND FLUORESCENCE The two-photon phenomena which have been described in this chapter are Rayleigh and, what has been called ordinary or normal, Raman scattering. In the treatment of these processes with the use of the semi-classical theory, it is necessary to invoke the concept of a hypothetical intermediate, or 'virtual' state of the molecule. The left-hand portion of the diagram shown in Fig. 6 (a and b) is usually employed, where the virtual states are indicated. It must be emphasized here that such virtual states do not exist and that these phenomena involve the simultaneous annihilation and creation of the two photons. Furthermore, as the intensity formula of Eq. (9) was derived with the aid of perturbation theory, it is not applicable when the light-matter interaction becomes strong. As an example of the limitations of the semi-classical theory, consider the case represented in Fig. 6c. Here, the frequency of the excitation is high enough so that its energy approaches those of the various excited electronic states of the molecule. Under these conditions, the second criterion for applicability of Eq. (9) fails. This result is obvious in that the second term approaches infinity as VQ-^ V^. This situation describes what is called near-resonance Raman scattering. Its mechanism is, at best, poorly understood. The cases represented by (d) and (e) in Fig. 6 are known as resonance Raman scattering and resonance fluorescence, respectively. In both of these phenomena the excitation is into the various vibrational energy levels of

///////z///////

1

//^

r\

n\

_ Si

3« w.

Excited electronic 0 states

Vir tUc l i s tat

c

\\

^t

\' _,.

\f

'f

]f

1f

^f 1

^f 1

Ground 2 electronic 0 states

Figure 6 Mechanisms of various light-scattering processes, (a) Rayleigh, (b) non-resonance Raman, (c) pre-resonance Raman, (d) resonance Raman and (e) resonance fluorescence.

The Raman Effect

21

the molecule in one or more of its excited electronic states. The distinction between these two effects, from both the experimental and theoretical points of view, has been discussed by many authors (see, in particular, Behringer, 1974, and references therein). In both cases scattered or emitted light is observed. However, the distinction between these two effects lies in the words 'scattered' or 'emitted', as employed in the previous sentence. In other words, the basic difference between these two processes depends on the time scales involved, as well as on the nature of the so-called intermediate state(s). In the case of the resonance Raman effect, and for the Raman effect in general, the entire process is completed within a picosecond or less. Resonance fluorescence, on the other hand, results from the emission of a photon from the lowest vibrational level of an excited electronic state, as shown in Fig. 6e. This emission is slow compared with the resonance Raman effect, requiring more than 10~^s in a typical case. There is thus sufficient time for the energy which is provided at excited vibrational levels to trickle down to the u' = 0 level of the particular excited electronic state. From a theoretical point of view, the intermediate state involved in a given resonance-fluorescence event is well defined and can be characterized by a lifetime TJ.. It is important to note that from Fig. 6d the frequencies of resonance Raman scattered Hght depend on VQ, the frequency of the excitation. On the other hand. Fig. 6e indicates that the frequency of fluorescence emission is independent of VQ. When VQ ~ Vj. resonance Raman scattering can occur. The semi-classical perturbation theory no longer applies and the scattering tensor is in general asymmetric. Thus, the antisymmetric invariant 2^, as defined by Eq. (13), is nonzero and the general expression of Eq. (20) must be used to determine the depolarization ratio appropriate to measurements on a liquid sample. For Raman fundamentals due to non-totafly symmetric vibrational modes, 2^ = 0 and /9>3/4 becomes possible. This effect is referred to as 'anomalous' polarization. Furthermore, in the limit as 2 ^ - ^ 0 , p approaches infinity. This situation gives rise to 'inverse' polarization, a phenomenon which has been observed in a certain number of cases. Similarly, with 2 ^ > 0 the totally symmetric modes ( 2 ^ > 0 ) can exhibit 'anomalous' polarization if In addition to resonance Raman and fluorescence, a third process can occur in which no radiation follows the absorption of a photon by the molecule. In this case the molecule returns to its initial state after collisions with other molecules or the walls of the container. The absorbed light energy is dissipated as heat. The analysis of resonance Raman spectra requires further theoretical considerations. As pointed out above, Eq. (9), which was originally proposed by Kramers and Heisenberg (1925), fails under resonance conditions. This problem was remedied by the rather artificial addition of a damping term

22

G. Turrell

iFj- in the denominators (Weiskopf and Wigner, 1930). Furthermore, near resonance the first term in Eq. (9) is negUgible with respect to the second, and a typical element becomes {oLxYh = TX

TTr

'

^26)

where F^ represents the width of the absorption band due to the transition to the electronic state r. This formalism was employed by Placzek (1934) in his discussion of resonance effects. It can be obtained directly with the use of quantum field theory (Hameka, 1965). It should be noted that the excited state wave functions such as |r) appear in the numerator of Eq. (26). This result is a consequence of the perturbation method employed in the derivation and does not imply that the Raman transition i ^ j occurs through a series of transitions i ^ r ^ j (see Eq. (8)). Therefore, the damping term cannot be related to a lifetime r^ via the expression F^ = \7rTr, as might be expected. In other words, as pointed out above, in the Raman experiment, in contrast to the fluorescence experiment, the molecule does not rest for a time in r^ an intermediate state r and the photon annihilation and creation are simultaneous events. Substitution of Eq. (26) into Eq. (8) provides an expression for a typical element of the scattering tensor. This result can be further developed to yield a basis for the interpretation of resonance Raman spectra (Albrecht, 1961). With the aid of the Born-Oppenheimer approximation, the function, which corresponds to the wavefunction for the vibronic state r, can be decomposed in the form k>=^e(^,G/c)^vib(e^)

(27)

for a particular vibration described by the normal coordinate Qx- The electronic part of the wave function is of course a function of both the electronic coordinates s of the molecule and the 3A^ — 6 normal coordinates Qj^. This function can then be developed in a Taylor series in the normal coordinates. Substitution of this result into Eq. (26), and, with the use of Eq. (8), leads to a general expression for an element of the scattering tensor in the form

({axY)K ^ = TIf/nl wi i m >V J1L^>M2)_ {0\iJ,x ir}{r\iJ-y\0} -—

VQ

+ iF^

The Raman Effect 23 Following Albrecht (1961), this equation can be decomposed into two parts, the 'A term'. (29) which is the first term of Eq. (28), and a 'B term', which is the sum of two essentially equivalent terms, the second and third in Eq. (28). This derivation involves several approximations which will not be discussed here. However, some general conclusions result from this analysis, e.g. significant enhancement of totally symmetric vibrational modes of a molecule occur when: (i) the absorption in an electronic band is strong; (ii) at least some product (1 |r)(r|0) of the Franck-Condon overlap integrals is numerically important; and, (iii) the exciting frequency VQ is close to the absorption band due to the electronic transition considered. This enhancement effect is attributable to the A term [Eq. (29)]. If the normal mode of vibration corresponding to the observed Raman fundamental is not totally symmetric, the first term in Eq. (28) vanishes, and resonance enhancement can only take place with the aid of the B term. In general, B term enhancement is significantly weaker than that arising from the A term. Thus, it is usually the totally symmetric modes of a polyatomic molecule that are susceptible to significant resonance enhancement. It is perhaps not appropriate to discuss here the question of what is referred to as the 'excitation profile' in resonance Raman spectroscopy. This term describes the intensity of Raman scattering as a function of the frequency of the excitation. While it might be assumed on intuitive grounds alone, that the excitation profile should correspond to the electronic absorption spectrum of the molecule, such is not the case. In fact, it has been shown that the shape of an electronic absorption band and the corresponding excitation profile are related by a Kramers-Kronig transform. The general theory of resonance Raman spectroscopy and, in particular, the calculation of the form of the excitation profile, is extremely difficult. The interested reader is referred to the work of Champion and Albrecht (1982).

XI. POSTLOG In this introductory chapter an attempt has been made to summarize those aspects of Raman spectroscopy which are directly involved in Raman microspectroscopic apphcations. The theoretical treatment of the subject, and, in particular, the mathematical developments, have been minimized.

24

G. Turrell

W h e n e v e r possible, basic references have been given in order to allow the interested reader to improve his or her basic understanding of the theory of the R a m a n effect.

REFERENCES Albrecht, A. C. (1961). /. Chem. Phys. 34, 1476. Anderson, P. W. (1949). Phys. Rev. 76, 647. Behringer, J. (1974). J. Raman Spectrosc. 2, 275. Bellamy, L. J. (1975). The Infrared Spectra of Complex Molecules. Chapman & Hall, London. Brawer, S. (1975). Phys. Rev. IIB, 3173. Brillouin, L. (1922). Ann. Phys. (Paris) 88, 17. Cabanes, J. (1928). Compt. Rend. Acad. Sci. Paris 186, 1201. Carey, P. R. (1982). Biochemical Applications of Raman and Resonance-Raman Spectroscopies. Academic Press, Toronto. Champion, P. M. and Albrecht, A. C. (1982). Ann. Rev. Phys. Chem. 33, 353. Damen, T. C , Porto, S. P. S. and Tell, B. (1960). Phys. Rev. 142, 570. Delhaye, M. and Migeon, M. (1966). Compt. Rend. Acad. Sci. Paris 262, 702; 1513. Delhaye, M. and Dhamelincourt, P. (1974). IVth Int. Conf. Raman Spectrosc, Brunswick, ME, USA. Dollish, F. R., Fateley, W. G. and Bentley, F. F. (1974). Characteristic Raman Frequencies of Organic Compounds. John Wiley & Sons, New York. Gordon, R. G. (1966). J. Chem. Phys. 44, 3083; 45, 1649. Gussoni, M. (1980). In: R. J. H. Clark and R. E. Hester (eds). Advances in Infrared Raman Spectroscopy. Heyden, London, ch. 2. Hameka, H. F. (1965). Advanced Quantum Chemistry. Addison Wesley, Reading, MA. Hirschfeld, T. (1973). / . Opt. Soc. Am. 63, 476. Kramers, H. A. and Heisenberg, W. (1925). Z. Phys. 31, 681. Landsberg, G. and Mandelstram, L. (1928). Naturwissenschaften 16, 557; 772. Long, D. A. (1977). Raman Spectroscopy. McGraw-Hill, New York. Loudon, R. (1964). Adv. Phys. 13, 423. Loudon, R. (1965). Adv. Phys. 14, 621. Maiman, T. H. (1960). Nature (London) 187, 493. Miyazawa, T., Shimanouchi, T. and Mizushima, S. (1958). J. Chem. Phys. 29, 611. Placzek, G. (1934). Rayleigh-Streuung und Raman-Effekt. In: E. Marx (ed.), Handbuch der Radiologic. Academische-Verlag, Leipzig, vol. VL2, p. 205. Porto, S. P. S. and Wood, D. L. (1962). /. Opt. Soc. Am. 52, 251. Raman, C. V. and Krishnan, K. S. (1928). Nature 111, 50. Raman, C. V. and Krishnan, K. S. (1929). Proc. Roy. Soc. Lond. Ill, 23. Rocard, Y. (1928). Compt. Rend. Acad. Sci. Paris 186, 1107. Rosasco, G. J., Etz, E. S. and Cassatt, W. A. (1974). IVth Int. Conf Raman Spectrosc, Brunswick, ME, USA. Rothschild, W. G. (1984). Dynamics of Molecular Liquids. John Wiley & Sons, New York. Schawlow, A. and Townes, C. H. (1958). Phys. Rev. 122, 1940.

The Raman Effect

25

Smekal, A. (1923). Naturwissenschaften 11, 873. Sonnich Mortensen, O. and Massing, S. (1980). In: R. J. C. Clark and R. E. Hester (eds), Advances in Infrared Raman Spectroscopy. Heyden, London, vol. 6. Stoicheff, B. (1963). X Colloquium Spectroscopicum Internationale, University of Maryland, June 1962. Spartan Books, Washington, DC. Turrell, G. (1972). Infrared and Raman Spectra of Crystals. Academic Press, London. Turrell, G. (1989). In: D. J. Gardiner and P. R. Graves (eds). Practical Raman Spectroscopy. Springer-Verlag, Berlin. Weiscopf, V. and Wigner, E. (1930). Z. Physik 63, 54. Wilson, E. B. Jr, Decius, J. C. and Cross, P. C. (1955). Molecular Vibrations. McGraw-Hill, New York.

Characteristics of Raman Microscopy George Turrell, Michel Delhaye and Paul Dhamelincourt

I. INTRODUCTION A general description of the Raman effect was presented in the first chapter of this book, and its apphcation to the analysis of microscopic samples was introduced from an historical point of view. In this second chapter those characteristics which differentiate Raman microspectroscopy from the more conventional techniques will be developed in more detail. The important characteristics of Raman microscopy are directly related to two fundamental optical considerations, namely: (i) the focusing of the incident laser excitation on the sample, and (ii) the collection of the scattered light. These aspects of the microscopic apphcation of the Raman effect will be treated in the following two sections. The specific problem of couphng a microscope to a Raman spectrometer is analyzed in Section V. Finally, in Section VI the confocal effect is described in some detail, as it forms the basis of recent advances in Raman instrumentation, including imaging techniques, which are presented in Chapter 4 of this volume.

II. EXCITATION FOCUSING In conventional Raman spectroscopy the exciting laser beam is usually focused on the sample with the use of a lens of 10-30 cm focal length. The

28

G. Turrell et a/.

laser light is then concentrated in a 'focal cylinder' (Long, 1977). A considerable gain in the intensity of Raman scattering is achieved by this process. It has been shown that the effect of focusing the laser beam does not in this case result in a significant depolarization of the excitation (Turrell, 1985). It should be pointed out, however, that the resulting higher irradiance may in some cases damage the sample being studied. When the laser beam is focused by an objective with a high numerical aperture the diameter of the focal region is ultimately determined by the diffraction limit, and hence by the wavelength of the light excitation. The resulting polarization of the excitation at the sample must be reconsidered. It has been shown by Richards and Wolf (1959) that the electric vector of the excitation in the focal region within a sample is given by ^i(/0 + /2( (1)

—±2 »iii Y

-211

COS if/

It has been assumed here that Z is the direction of propagation of the laser beam and that it is plane-polarized in the X direction before passing through the microscope objective. The angle if/ is measured in the counterclockwise sense with respect to the X axis. The integrals involved in Eq. (1) have been derived for the case of a nonabsorbing, isotropic sample of refractive index n in the form (Bremard et al., 1987a) Io(u,v,n)

=2

D{e)sme Jo X cos OJQ

/i(w,u,n) =2

1 m -h n^ cos ^ -f m cos 6-\-m

usin^ sin^^

D(e)sine JO

exp(iw cos ^/sin ^^) cos^^^ ^d^,

sin^ cos 6 J n^ cos 6-^ m

(2)

vsinS sin Srr,

X exp(iw cos 0/sin^ 6^) cos^^-^OdO

(3)

and OrTi

/2(w,u,n) =2

D{e)sine Jo xcos^72

m n^ cos 0 + m

usin^ sin^^

1 cos d-\- m

exp(iw cos O/sin^ 0^) cos^^^ddO,

(4)

which 6 is the angle of incidence of a given ray of the light excitation, D{6) = A^csc^exp(—sin^^/sin^^^), which represents a Gaussian radial distribution in the laser beam, and A'^ is a normalization constant. A point in the focal region is specified by the dimensionless cylindrical coordinates

Characteristics of Raman Microscopy 29 u = kZ^iv?'d^ and v = k{X^ + Y^Y'^ sin 6^, as well as the angle ifj. Here k = lirlX is the wave number, m = V(n^ - sin^ 6) and the J^s are the Bessel functions of the first kind. The integrals defined by Eqs (2-4) are functions of n, the real, isotropic refractive index of the sample.

III. COLLECTION OPTICS Equation (7) of Chapter 1 shows that the total Raman intensity depends on the solid angle O in which the scattered Ught is collected. In the backscattering configuration fl describes approximately the cones of both the incident and scattered hght. When the cone axis is coUinear with Z and the excitation is assumed to be polarized in the X direction, the expression for the relative Raman intensity becomes (Turrell, 1989) 3 = (aj,xA + aj^A

+ alzB)(2Co

+ C2)

+ (ai^x^ + ctYY^ + oti^zB) C2 + (alxA

+ alyA

+ a|z5)4Ci,

(5)

where

J

'*oo roo

\Ij(u,v,n)\^vdvudu,

j =0,1,2,

(6)

0 Jo The parameters A and B, which are obtained by integration over the scattering cone, are defined by re'm /^ 1 \ A = 7T^\ (cos^ ^ + 1) sin 6> d^ = 77^ - - cos 9'^-cos^ 0'^]

(7)

re'm 12 1 \ sin^ ^ d^ = 277^ - - cos ^;„ + - c o s ^ d'rn \

(8)

and ^ = 277^

w h e r e ^ ^ is the effective half angle of the cone. These p a r a m e t e r s are plotted as functions of n in Figs 1 and 2, respectively. T h e values of the integrals Cj are presented in Figs 3-5 as functions of the index of refraction, n. E q u a t i o n (5) serves as the basis for the interpretation of polarization m e a s u r e m e n t s on isotropic media. If the scattered Hght is analyzed in the direction {X) parallel to that of the polarization of the excitation, the intensity of the scattered light is given by

3|| = {a\xA

+ a\zB) (2Co + C^ + {a\xA

+ {alxA-^alzB)^C^.

+ a\zB) C^ (9)

30

G. Turrell et a/.

3

2

Refractive index, n

Figure I

Parameter A as-afunction of refractive index.

On the other hand if the analysis is perpendicular to the direction of polarization of the excitation, the expression for the intensity becomes 3 ^ = (aj^yA + aj,zB){2Co + C2) + {al^yA + a^yzB) C2 + {alyA

+

alzB)AC,.

(10)

Thus, the nonzero values of 5 , Ci and C2 can be used to evaluate the 'polarization leakage' which is observed in the Raman spectra of single-crystal samples. This problem has been recently summarized (Turrell, 1989). For gaseous and liquid samples the intensities of the scattered light are

Characteristics of Raman Microscopy

31

10

2

3

4

Refractive index, n

Figure 2 Parameter fi as a function of refractive index.

then given in terms of the tensor invariants X*', S^ and 2^ [Eqs (12-14) Chapter 1] by 3|| = [\A1P

+ iSS^ + {U

+

TOS)22]2CO

+ [152° + \AV + iU + ^5)22]4Ci + [M2« + {\A +\B)X^ + i^A + \B) 22] 4C2

(11)

and 3_L=(|2i + Ji22)(^ + B)2Co + [i522 + iA2i + {^oA + *5)22]4Ci + [1^2" + (lA +15) 21 + (^^ + IB) 22] C2,

(12)

respectively. The depolarization ratio defined in Eq. (19) of Chapter 1, by p = 3jy3|| is then calculated from these relations. Equations (9) and (10) have been employed in the interpretation of Raman spectra of both isotropic and anisotropic samples. In the case of isotropic

32

G. Turrell et a/.

Co

2

3 Refractive index, n

Figure 3 Parameter Q as a function of refractive index.

samples correct depolarization ratios can be obtained even when objectives with high numerical aperture are used. However, as a beamsplitter is usually included in the optical system, its transmission characteristics must be evaluated and appropriate corrections introduced (Bremard et al., 1985). If the sample is optically anisotropic, the analysis is considerably more complicated. However, the depolarization effects introduced by the wideangle objectives are not large. They can be evaluated theoretically if the birefringence of the sample is negligible (Bremard et al., 1987b). The depolarization due to highly convergent incident and divergent scattered light is especially important if propagation is in a direction close to that of an optical axis of a crystalline sample. This effect can be minimized by reducing

Characteristics of Raman Microscopy

1

2

33

3

Refractive index, n

Figure 4 Parameter Ci as a function of refractive index. the optical path (depth of focus) within the sample (Bremard et al., 1989). The apphcation of the above analysis in resonance Raman spectroscopy has also been demonstrated (Bremard et al., 1986, 1987a). In this case the nonvanishing of the tensor invariant S^ often results in so-called inverse polarization, in w^hich p becomes very large (cf. Chapter 1, Section X).

IV. ABSORBING SAMPLES

The geometrical problems associated v^ith sample illumination and the collection of Raman-scattered hght were analyzed in the two previous

34

G. Turrell et al.

2

3

4

Refractive index, n Figure 5 Parameter C2 as a function of refractive index.

sections. However, the sample was assumed to be transparent, i.e. nonabsorbing at both the excitation and scattering frequencies. The case of absorbing samples, which has not as yet been sufficiently studied, presents certain practical difficulties. For the traditional 90° configuration, when employed in the observation of Raman spectra of solutions, it has been demonstrated that there is an optimum concentration. Thus, the relative Raman intensity as a function of concentration displays a maximum (Strekas et al., 1974). A simple model is qualitatively consistent with this observation, although quantitative agreement with the experimental measurements has not been obtained (Renaut et al., 1988). It was shown many years ago by Hendra (1967) that useful Raman spectra of very opaque materials such as coal could be obtained in the backscattering configuration. As it is this geometry that is employed in micro-Raman spectroscopy, this observation has become extremely important. Thus, the

Characteristics of Raman Microscopy

35

OBJECTIVE

/

/

/

/

EXCITATION SCATTERING

I--Figure 6 Backscattering geometry.

problem which is presented by strongly absorbing samples, as observed with a Raman microprobe, will be briefly summarized here. The analysis (Turrell, 1989) is made on the basis of the geometry shown in Fig. 6. The laser excitation is assumed to consist of a parallel beam within the focal cyUnder. For a Gaussian radial distribution in the exciting beam, the irradiance at a point within the sample is given by 4 = /e^exp(-C8eZ) exp[-4(Z2 + Y^)ld^].

(13)

The intensity of the light which is scattered by a volume d X d Y d Z within the sample can be expressed in the form d3^a4cdZdydZ,

(14)

and the intensity of the scattered light leaving the sample in the Z direction is then d3s = d3^exp(-c£sZ).

(15)

36

G. Turrell et al.

H ^

8 = 2 X 10'

5

< H

Z ^

2

Figure 7 Raman scattering intensity plotted against concentration (c) of absorbing solutes.

Equations (14) and (15) lead to the relation d% oc /Oc exp [-c(8s + 8^)Z] AZ exp [ - 4 ( ^ 2 + Y^)ld'^] dXdY,

(16)

whose integration yields approximately 3s--

7 r ( l - l / e ) I^d^ ( l - e x p [ - c ( £ e + £s)^])16 e^ + e.

(17)

Here, L is the effective depth of focus (see Fig. 6). This result is plotted in Fig. 7, where it is seen that for a given value oi e = E^-\- e^ that the relative Raman intensity does not decrease at high concentrations. This result, which has been obtained on the basis of a simple model, has been at least semi-quantitatively confirmed by direct measurement (Renaut, 1988). A problem which arises from sample absorption is their degradation under intense laser Hght excitation. This situation is characteristic of the Raman microprobe. The 'burning' of specimens is certainly due to a combination of thermal effects and photochemical decomposition. However, it is a problem which has not as yet received sufficient attention, although it is a very serious one in Raman microscopy.

Characteristics of Raman Microscopy

37

V. MICROSCOPE-SPECTROMETER COUPLING This section will be devoted to the fundamental problems, as well as the practical design, of the coupling optics between a microscope and a spectrometer or spectrograph.

A. Coupling Conditions As will be shown in the following chapter, the optimum use of the Raman light flux ^ collected from a sample requires that it be transmitted from sample to detector via the successive apertures of the instrument. In general, microscope objectives are aplanetic, so that the rule for maintaining the flux (assuming perfect transmission of all optical elements) is to apply the Abbe invariant n^h-^^m^i (see Fig. 8) at each intermediary image of the sample, as well as at each intermediary aperture along the optical path of the instrument. The Abbe invariant rule ^o^o sin ^o = ^\^'\ sin ^ i . . . = ^o/io sin ^o is indeed equivalent to the conservation of the optical extent f/f (see Chapter 3) of the Raman light beam passing through the instrument without loss in flux. This condition can be expressed by multiplying the square of the Abbe invariant by TT. This operation leads to the expression n^TTS^ sin^ ^o = • • • ^"^Sx sin^ ^j = . . . noTr^o sin^ B[ =

(/)/LR,

(18)

where 5-0, s^ and 5o are the areas of the sample, of any intermediary image of the sample and of the image of the sample from or to the entrance sUt of the spectrometer, respectively. In Eq. (18) L R is the brightness of the Raman source at the sample.

MICROSCOPE AND COUPLING OPTICS

SPECTROMETER

Figure 8. Application of the Abbe invariant rule for each intermediary image and pupil throughout the optical path.

38

G. Turrell et al.

B. Design of Coupling Optics The invariance conditions of the optical extent can be fulfilled with the use of coupling optics which result in good matching between all of the apertures along the entire hght path, from microscope to the spectrometer detector. The matching of the apertures means that the coupling system not only has to conjugate optically all of the intermediary images of the sample with the entrance slit but, at the same time, it must conjugate all of the intermediary apertures with the entrance pupil of the spectrometer. Assuming that in a spectrometer the acceptance angle ^o of the first monochromator is small, the following expression can be written (see Fig. 8), sme()^e()--p/2D,

(19)

where p is the effective size of the grating pupil of the monochromator and D the distance between the pupil and the slit. The Abbe invariant noho sin ^o = ^6 sin ^o can be rewritten in the form 7o

2D

where (N. A.) and y^ are the numerical aperture and the magnification factor of the microscope objective, respectively. The magnification factor of the coupling optics is given by y^. In this formula the inequality expresses the result that the pupil of the monochromator must not be overfilled by the light diffused by the sample. The ratio p/D, which is characteristic of the monochromator aperture, is constant for a given instrument. It is then clear that the magnification factor of the coupHng optics is directly related to the characteristics of the microscope objective. There are then several possibiUties for coupling microscopes with spectrometers. They can be summarized as follows. (i) Use an adjustable zoom for any particular objective mounted on the microscope turret, at the expense of poor transmission. (ii) Design as many interchangeable fixed magnification optical systems as needed for all of the objectives mounted on the microscope turret. (iii) Design a unique optical system whose magnification is adapted to a particular objective and which is suitable for insuring both good spatial resolution and maximum collection of Raman light. Generally, this condition is applied to objectives with high N. A. and high magnification, the others mounted on the microscope turret being chosen according to the (N.A.)/yo restriction given by Eq. (20). The last possibility is, in practice, the best one. Indeed, fixed magnification coupling optics are easy to design and have an excellent transmission factor

Characteristics of Raman Microscopy

39

( r > 0 . 9 ) , when constructed with coated lenses. Several lens combinations are possible. A simple three-lens system, which was designed for the first MOLE instrument (Dhamelincourt, 1979, 1982), is presented in Fig. 9 as an example. Ray tracing indicates clearly both the optical couphng of the image of the sample with the entrance sUt and that of the aperture with the entrance pupil of the spectrometer.

MICROSCOPE

COUPLING OPTICS

Figure 9. Example of a three-lens, optical-coupling system. The ray tracing illustrates the matching of the microscope objective and the spectrometer apertures.

VI. CONFOCAL RAMAN MICROSCOPY A. Introduction The concept of confocal scanning microscopy was introduced by Minsky (1988) in the early 1960s to overcome some of the limitations of the conventional optical microscope. With this technique a significant improvement in both the contrast and the spatial resolution may be obtained when a point source is focused at the diffraction hmit onto the specimen, while the enlarged image of the illuminated spot is analyzed through a pinhole diaphragm. Unlike a conventional microscope, where the entire field is illuminated, the confocal system measures at any one time the intensity of the light reflected or transmitted by a very small area of sample. A reconstitution of a two-dimensional image is performed via numerical data treatment of the photoelectric signals resulting from a sequential analysis by an XY-raster scanning of the sample field. A three-dimensional display may also be produced by combining a series of optical sections recorded sequentially by means of a motorized Z-focus attachment. Detailed theoretical and experimental studies of the properties of confocal microscopes may

40

G. Turrell et a/.

be found in books by Wilson and Sheppard (1984, 1989) and Wilson (1990). Without entering into complicated calculations, the essential features of confocal microscopy may be summarized as follows: (i) An exact optical conjugation onto the sample of the pinhole apertures which are employed for both illumination and detection of the specimen results in combined spatial filtering effects. They produce a narrower 'point spread function' than can be obtained with a conventional microscope. (ii) The stray light background due to the out-of-focus regions of the specimen is strongly attenuated by spatial filtering, so that the main contribution to the signal comes selectively from a thin layer of sample close to the exact focal plane. This capability of optical sectioning undoubtedly constitutes the most important advantage of the confocal configuration, which benefits from both the contrast enhancement and the improved depth of field. The application of the confocal principle to Raman spectroscopy is not straightforward. In fact the first experimental Raman microprobes, conceived and developed in the early 1970s (Delhaye and Dhamelincourt, 1975; Rosasco, 1980), effectively employed two conjugated spatial-filtering diaphragms, one for laser excitation and the other for measurement of Raman scattered light. However, the inherent weakness of Raman signals and the lack of sensitivity of the existing photodetection systems available at that time did not allow the reduction of the pinhole diameters to achieve the diffraction limit. Consequently, the conditions required to obtain optical sectioning with a micrometer axial resolution were not fulfilled. Another limitation, which is particularly severe for Raman microprobing, originates from the very intense laser radiation which is, for the most part, elastically backreflected by the specimen. Unlike the usual scanning, optical microscopes, a Raman instrument must necessarily discriminate between the weak spectral signals and the signal due to the nearby extremely strong excitation radiation. Now, almost 20 years after the appearance of the first prototypes of Raman microprobe instruments, especially designed and optimized systems which benefit from the dramatic improvements in multichannel photodetectors and spectral filtering, have been described (Puppels et al., 1991; Tabaksblat et al., 1992; Barbillat et al., 1992a; Dhamelincourt et al., 1993). Unlike the first generation of Raman microspectrophotometers in which most instrument makers simply added a microscope attachment to an existing monochromator, the commercial instruments which are now available provide the long-term accuracy and stabihty of the optical adjustments that are absolutely necessary to obtain high spatial resolution and depth

Characteristics of Raman Microscopy

41

EYEPIECE

COUPLING OPTICS D2

I

REMOVABLE MIRROR I

PINHOLE SPATIAL FILTER

PINHOLE SPATIAL FILTER

SPECTROMETER ENTRANCE SLIT

BEAMSPLITTER

MICROSCOPE OBJECTIVE

SAMPLE

Figure 10. Schematic diagram of the laser focusing, sample viewing and scattered light collection geometry which is widely employed in micro-Raman spectrometers. discrimination (DILOR, 1991; Delhaye et aL, 1992; Barbillat et al, 1992b, 1994; Manfait et al, 1992). The main features of a Ramann confocal system can be described by the basic layout shown in Fig. 10. The laser excitation beam is first filtered by an illumination pinhole Di. This initial spatial filtering removes the appearance of diffraction rings and speckle noise around the focused spot and results in a clean point source waist which is imaged onto the sample. The scattered Raman radiation is collected by a wide-aperture objective and focused on an adjustable pinhole D2 placed in the image plane of the microscope. A beamsplitter insures a coaxial illumination and Hght collection by the same objective in the backscattering configuration. The pinholes Di and D2 are called confocal diaphragms. Their exact optical conjugation with the point source in the object plane ensures that only the light originating from the sample region, which coincides exactly with the illumination spot, is transmitted to the spectral analyzer and detector. The two effects of spatial filtering, both for illumination and collection, multiply and increase the spatial resolution by ehminating stray light coming from the out-of-focus regions of the sample. This configuration accounts for the ability of the system to provide optical sectioning. In Fig. 11 the variation of the signal intensity is plotted as a function of the axial coordinate Z, when a thin layer of sample is displaced along the

42

G. Turrell et al. J PHOTODETECTOR SIGNAL

AXIAL DISTANCE PHOTODETECTOR

I CONJUGATED PINHOLE / 1 A- DIAPHRAGM

/ BEAMSPLITTER

LASER BEAM

/ ^I 7i^-^-AZ / / ^ Y // .*^^ 'f^^ --'•/•--i__/

FOCAl.PLANE . i' z =o

i

i

1^

-^-

I

^

MICROSCOPE OBJECTIVE THIN SAMPLE SLICE

Figure 11. Effect of sample position on the photodetector signal in the confocal optical configuration.

optical axis. The axial resolving power, or depth of focus, is usually defined as the full width at half maximum (FWHM) of this curve. Decreasing the size of the confocal diaphragm improves both the axial resolution and reflection of stray light. In order to offer maximum versatility of the system, modern confocal Raman microscopes are provided with variable aperture pinholes, which enable an optimum adjustment to be made of the recording conditions. The magnification factor of the microscope may be varied, usually by means of interchangeable objectives mounted on a turret. It is worth noting that optimal performance requires a correct matching of all of the apertures and pupils along the entire light path, both in the microscope and in the spectral analyzer section (see Section V.B). Improper beam matching severely limits the capabilities of the instrument. Consequently, the coupling optics which have been designed by the instrument maker are only optimized to fulfill the matching conditions for certain objectives, and for the beam divergence for a given laser excitation. In particular, overfilling or underfilling of the microscope objective by the laser beam modifies the dimension of the focused spot, as well as the radiance distribution, at the expense of the spatial resolution and/or the signal intensity. The critical importance of a correct adaptation of all optical parameters will be emphasized in the next sections of this book. The numerous applications of confocal Raman microprobing and microscopy will be described and references to the appropriate literature will be given.

Characteristics of Raman IVIicroscopy

43

B. The Confocal Effect

The light scattered by the Raman effect (or fluorescence) is incoherent in nature and, therefore, can be treated with the use of conventional optical laws, assuming that diffraction effects are weak. 1. Raman Light Flux Emitted by a Thin Slice of Sample Consider, in the object plane, a very thin slice of an isotropic and homogeneous sample whose surface SQ is defined by the waist of the focused laer beam (Fig. 12). Assuming a radial profile for the laser excitation, this slice can be considered to be a Raman light source emitting uniformly in all directions. The elementary Raman light flux captured by the objective for a backscattering angle 0 and an elementary solid angle dfi can be expressed as d^^Q =

(21)

L^dH,

where L R and ^% are the Raman source luminance (brightness) and the geometric etendue or throughput of the elementary Raman light tube, as defined by 6 and dfl, respectively. dop/odz, dil

LR =

(22)

where (dcr/dfl) is the differential Raman cross-section for a given band and a given exciting wavelength, p is the molecular density (number of molecules per unit volume), /Q is the laser irradiance at the sample (power per unit area) and dz is the slice thickness. The geometrical extent is given by d2^ = d5Cos^dn,

SAMPLE OBJECT SLICE PLANE

OBJECTIVE-TUBE LENS COMBINATION

BEAM SPLITTER

1

ENTRANCE / EXIT PUPIL ^ PUPIL LASER SOURCE

(23)

IMAGE PLANE

ENTRANCE PUPIL OF COUPLESfG OPTICS

Figure 12. Collection by the microscope objective-tube-lens combination of the Raman flux from a sample slice.

44

G. Turrell et a/.

where ds is the elementary surface around one given point of the surface slice, and 6 and dft have been defined above. The total flux entering the instrument is then

dH,

ds

do = ^ R * ^ = ^ R 0

(24)

0

where ^o is the half-acceptance angle of the microscope objective. Assuming now that dz and 5*0 are small compared to the working distance and the frontal lens diameter, respectively, % may be written in the form ds

sin ^ cos ^d^.

(25)

Thus, after integration d(/)o = L^TTSQ sin^ ^o-

(26)

The Raman light flux entering the instrument is proportional to the square of the numerical aperture of the objective (N.A. = n sin OQ, where n is the refractive index of the object-space medium). Therefore, a significant gain in the light collection efficiency for a thin sample can be achieved with the use of objectives with large numerical aperture. 2. Transmission of the Raman Light Flux through a Confocal

Diaphragm

In the image space, the application of Clausius's law to the combination of objective-tube lens, which is equivalent to the condition for the invariance of the optical etendue of the Raman light tube, yields an expression for the flux entering the pupil of the coupling optics as follows: d<j>'o = TL'^%\

(27)

where r is the transmission of the objective-tube-lens combination, L R = L R M ^ (Kirchoffs law) and %' = 7TSQ%\V? OQ. Here SQ and ^o represent the surface of the image of the slice and the half angle subtended by the marginal ray entering the pupil of the transfer (couphng) optics, respectively. Thus, d<^o = ;^(-^)p/odz7756sin2^6,

(28)

which may be rewritten in the form dcf>'o = /o5o| - ^ 1 p d z - ^ l ^ ) sin^ dl,.

(29)

Introducing P^, the power at sample, the magnification factor of the

Characteristics of Raman /\/!icroscopy OBJECT PLANE

45

IMAGE PLANE

OBJECTIVE-TUBE LENS COMBINATION

SAMPLE SLICE

Figure 13. Ray tracing illustration of the loss offluxthrough the confocal diaphragm for a sample slice outside of the object plane of the objective.

objective-tube-lens combination JQ, and considering that ^o is always small, finally yields the relation TTT

9

,2LI instrument

sample

(30)

where cf)^ and L are the diameter of the entrance pupil of the transfer optics and the distance between this pupil and the image plane of the objectivetube-lens combination, respectively. Thus, thefluxentering the coupUng optics comprises two terms, which are the luminous intensity of the source (Raman scattering by the sample sUce) and the throughput of the objective-tube-lens combination, respectively. However, this expression is valid only when the confocal diaphragm D2 is of a dimension which is at least equal to that of the image of the sample slice produced by the objective-tube-lens combination. When the sample slice is not in the object plane, only a part of the light which passes through the confocal diaphragm D2 is transmitted to the coupling optics. In this case the flux has to be re-evaluated as follows. In the following formulae upper and lower signs have been employed in order to simplify the notation. However, only one sign is valid, depending on the position of the sample slice (see Fig. 13) and the flux is then given by

d06 = A

TTT

dfi r

9 /

(l)c/2

(31)

where y^ is the magnification factor of the objective-tube-lens combination when the sample slice is at a distance A from the object plane. The distance between the slice image and the image plane is given by A' and Tp is the transfer function of the confocal diaphragm Di.

46

G. Turrell et a/.

The function Tp can be expressed in the form Jo°'E(R)ds ^ = :Vc:^ ^ jt^EiR)ds

>

(32)

where i?Lc ^^^ ^ D I ^re the radii of the luminous circle formed in the image plane and of the diaphragm D^, respectively. The illumination distribution in the image plane is represented by E{R). From simple geometrical considerations the relation (c^ex/2)+i^LC ^

(ex/2) + ^^

P

P ± A'

^ ^

rX is the radius of the image slice and can be obtained, where p is the distance between the exit pupil of the objective-tube-lens combination and the image plane. Considering that (/)ex/2 = PfJ^o the relation ^LC = ^ ^ ( ^ 6 A ' + ri)

(34)

/? ± Z\

is obtained, where 0ex is the diameter of the exit pupil of the objective-tubelens combination. Applying the Abbe sine condition to this combination yields the expression ^^^.sin^o^(RAO To

^35^

To

which in turn leads to

The application of the magnification laws corresponding to the objectivetube-lens combination r^ = y^r^, A' = JAJO^ and ( I / ^ A ) = (l/7o) - A//, where / is the focal length of the combination, yields the relation _ y J ( N . A . ) A + /-A] "-^

J-TATOA

•

(37)

P Here, r^ is derived from the focusing law for a Chapter 3) within an isotropic medium, /-A = roV[l + {^o^f],

TEMQO

laser beam (see (38)

where r^ = 0.61A/(N.A.) is the radius of the waist of the focused laser beam

Characteristics of Raman Microscopy

47

inside the sample and A is the laser wavelength. Finally, the flux entering the pupil of the coupling optics may be rewritten as

thus, d
(^)W

(40)

where the ratio i?(A) = ^ do

(41)

is the basic expression of the confocal effect. The axial resolving power, which is the instrument's ability to discriminate between parts of the sample which are not at the same depth, is defined as the full width at half maximum of the curve obtained when i?(A) is plotted against A. Two cases have to be considered depending on Dj, the aperture of the confocal diaphragm. (i) If i?Di-^LC? all of the Raman light passes through the confocal diaphragm (7^= 1). Then, in this case

Thus, /?(A) = 1 if A = 0 and it stays very close to unity when A varies. No confocal effect is observed. (ii) If i?D^ < i?Lc? only part of the Raman light is accepted by the coupling system ( r F < 1). Here, two possibihties have to be considered depending on the illumination distribution in the image plane. In the case of uniform illumination E{K) is constant and Tp = (i^o/^Lc)^This result leads to the relation

W - I -^^r^ I [..uJ^L^.A .

(43)

However, the assumption of a Gaussian distribution is probably the best

48

G. Turrell et a/.

model to describe the illumination in the image plane by a Raman source which obeys Lambert's law. In this case E{R) = £n.ax exp[-/:(/?//?Lc)'],

(44)

where k determines the width of the Gaussian curve. Then, J ^ Jo'"'^maxcxp[-k(R/Ri^cf]2iTRdR ""

fo'^^E^,,cxp[-k(R/Rj^cf]2iTRdR

^ 1-

exp[-k{Rr,JRi^cf] l-exp(-/:)

Thus, for a given objective, the axial resolving power will decrease rapidly as the aperture of the confocal diaphragm increases, the optimum axial resolving power being achieved when the size of the aperture of the confocal diaphragm is equal to that of the image of the laser waist produced at the sample by the objective-tube-lens combination.

C. Conclusion The confocal configuration offers the possibility of 'optical sectioning' of a sample. A variable confocal effect can be obtained by changing both the opening of the confocal diaphragm and by choosing the couple (magnification, numerical aperture) of the objective. This procedure allows optimum adjustment to be made of the operating conditions, depending on the sample geometry. Thus, when an isotropic, thick sample is examined, a large opening of the confocal diaphragm Di is recommended. Then, all of the Raman light flux originating from the probed volume is transmitted to the detector. However, when thin or heterogeneous samples are examined the opening of the confocal diaphragm has to be carefully adjusted so that only the Raman flux originating from the interesting part of the sample is transmitted to the detector.

REFERENCES Barbillat, J., Da Silva, E., Delhaye, M., Dhamelincourt, P., Manfait, M. and Roussel, B. (1992a). In: W. Kiefer (ed.), Proc, Xlllth Int. Conf. Raman Spectrosc. John Wiley & Sons, Chichester, p. 1074. Barbillat, J., Delhaye, M. and Dhamelincourt, P. (1992b). In: G. W. Bailey, J. Bentley and J. A. Small (eds), Proc. 50th Ann. Meet. Electron Microsc. Soc. America. Electron Microscopy Society of America, Woods Hole, MA, p. 1514. Barbillat, J., Da Silva, E., Lenain, B., Manfait, M., Sharonov, S. and Valisa, P. (1994). In: N. T. Yu and X. Y. Li (eds)., Proc. XlVth Int. Conf. Raman Spectrosc. The Hong Kong University of Science and Technology, Hong Kong, p. 1102. Bremard, C , Dhamelincourt, P., Laureyns, J. and Turrell, G. (1985). Appl. Spectrosc. 39, 1036.

Characteristics of Raman Microscopy

49

Bremard, C , Dhamelincourt, P., Laureyns, J. and Turrell, G. (1986). / . Mol. Struct. 142, 13. Bremard, C , Laureyns, J., Merlin, J.-C. and Turrell, G. (1987a). / . Raman Spectrosc. 18, 305. Bremard, C , Laureyns, J. and Turrell, G. (1987b). Can. J. Spectrosc. 32, 70. Bremard, C , Laureyns, J. and Turrell, G. (1989). / . Chim. Phys. 86, 1247. Delhaye, M. and Dhamelincourt, P. (1975). /. Raman Spectrosc. 3, 33. Delhaye, M., Da Silva, E. and Barbillat, J. (1992). European Patent no. 92 4001415. Dhamelincourt, P. (1979). In: D. E. Newbury (ed.), Microbeam Analysis. San Francisco Press, San Francisco, p. 154. Dhamelincourt, P. (1982). In: K. F. J. Heirich (ed.), Microbeam Analysis. San Francisco Press, San Francisco, p. 261. Dhamehncourt, P., Barbillat, J. and Delhaye, M. (1993). Spectrosc. Europe 5, 16. DILOR (1991). Unpublished technical documentation. Hendra, P. J. (1967). / . Chem. Soc. A, 1298. Long, D. A. (1977) Raman Spectroscopy. McGraw-Hill, New York. Manfait, M., Riov, J. F., Morjani, H., Lavelle, E. and Nabiev, I. R. (1992). In: W. Kiefer (ed.), Proc. Xlllth Int. Conf. Raman Spectrosc. John Wiley & Sons, Chichester, p. 520. Minsky, M. (1988). Scanning 10, 128. Puppels, G. J., Colier, W., Olminkhof, J. H. F., Otto, C , de Mul, F. F. M. and Greve, J. (1991). / . Raman Spectrosc. 22, 217. Renaut, D. (1988). Diplome d'Etudes Approfondies, University of Lille. Renaut, D., Turrell, S., Merlin, J.-C. and Turrell, G. (1988). In: Clark, R. J. H. and Long, D. A. (eds), Proc. Xlth Int. Conf. Raman Spectrosc. John Wiley and Sons, Chichester, p. 717. Richards, B. and Wolf, E. (1959). Proc. Roy. Soc. Lond. A253, 358. Rosasco, G. J. (1980). In: R. J. H. Clark and R. E. Hester (eds). Advances in Infrared and Raman Spectroscopy. Hey den, London, vol. 7, pp. 223-282. Strekas, T. C , Adams, D. H., Packer, A. and Spiro, T. G. (1974). Appl. Spectrosc. 28, 324. Tabaksblat, R., Meier, R. J. and Kip, B. J. (1992). Appl. Spectrosc. 22, 217. Turrell, G. (1985). / . Raman Spectrosc. 15, 103. Turrell, G. (1989). In: D. J. Gardiner and P. R. Graves (eds). Practical Raman Spectroscopy. Springer-Verlag, Berlin, pp. 13-54. Wilson, T. (1990). Confocal Microscopy. Academic Press, London. Wilson, T. and Sheppard, C. (1984, 1989). Theory and Practice of Scanning Optical Microscopy. Academic Press, London.

Instrumentation Michel Delhaye, Jacques Barbillat, Jean Aubard, Michel Bridoux and Edouard Da Silva

I. INTRODUCTION

The purpose of this chapter is to review the main features of the different parts of a modern Raman microspectrometer, including the basis of data acquisition and processing. It is important to emphasize that the instrument has to be conceived as whole, so that its overall performance is determined by an optimized combination of all of its components. To accompUsh the goal of obtaining the best possible spectral and spatial data, each component has to be designed or selected to fit into an integrated unit which insures maximum reUabiUty and stability of optical alignment and mechanical adjustment. The dominant factors which must be considered are: (i) Quality, filtering and focusing of the laser excitation beam; (ii) Wide soUd angle of collection with minimum aberration; (iii) Optimized optical coupling of the observed microvolume of sample to the photodetector via the spectral analyzer; (iv) Efficient rejection of stray light due to specular or diffuse reflection of the excitation radiation, which would otherwise degrade the signal-tonoise ratio (S/N); (v) High quantum efficiency and detectivity of the photodetectors usually employed in the shot-noise-Hmited regime; and (vi) Sophisticated data accumulation and processing of the spectral and spatial information.

52

M. Delhaye et a/.

II. CHARACTERISTICS OF LASER SOURCES A. Characteristics of a Gaussian Beam Because of its importance in the present application, some characteristics of a Gaussian laser beam will be summarized. A laser operating in its fundamental (TEMQO) mode can be assumed to emit such a beam, that the electric field is a Gaussian function of the radial distance r in the planar cross-section of the beam. In this case the minimum beam waist is of diameter 2WQ and the wavefront is planar at this point (see Fig. 1). In a particular application the minimum waist position may be in the laser cavity, at its exit or outside the cavity. The Gaussian description of the laser beam was used in Chapter 2, Section II. The radial distribution of the electric field and the irradiance in a Gaussian beam are given by %(r) = %oQxp{-r^/w^)

(1)

I{r) =

(2)

and Ioexp(-2r^/w^),

respectively, where w is the radial distance at which the electric field drops to 1/e of its axial value. The quantity w is the beam radius; thus, by integrating the irradiance over the beam diameter it is found that approximately 86% of the light falls within the spot. The beam diameter and wavefront curvature at a distance z from the minimum waist position are given by 2w{z) = 2woV[l +

(3)

(Z/ZR)2]

and /?(Z) = Z[1 + (ZR/Z)2],

(4)

^(z)

Figure 1 Geometry of a circular Gaussian beam.

Instrumentation

53

respectively. The quantity ZR = 7T{2W^^IAX is known as the Rayleigh range of the beam. The full, far-field divergence of the beam can be expressed as ^n =

2w{z)

— ' = —•;:— = 1 . 2 7 - — , 77 2Wo

ZR

(5)

2WQ

Note that for a plane wavefront incident on a circular aperture of diameter D, the full-cone angle defined at the first minimum of the Fraunhofer diffraction pattern (central Airy disc) becomes equal to ^F = 2.44A/Z).

B. Lens-focusing of a Gaussian Beam The effect of focusing a Gaussian beam by a thin positive lens is shown in Fig. 2. Here, 2WQ and 2wi are the beam waists of the real object and image, respectively. The circular Gaussian beam is transformed into another circular Gaussian beam by the lens. It has been assumed here that the pupil of the lens (of diameter 2w^) is correctly matched to the beam diameter. With the use of Eq. (3) the spot diameter 2wi at the focal point, at distance s' from the lens, can be evaluated, since 2w^= 2wi

-0

1/2

(6)

with ZR = 7r(2wi)^/4\. The far-field divergence is now given by ^i = 1.27A/2wi, where it should be noted that 2wi0i =

2WQ6O.

Figure 2 Lens focusing of a Gaussian beam.

(7)

54

M. Delhaye et al.

If the reasonable assumption is now made that s' « / , where / i s the focal length of the lens, ^i = 2Hy/=1.27A/2wi

(8)

2WI = 1.27A//2HV.

(9)

or Note that a more rigorous development is made below. However, for practical purposes Eq. (9) yields 2wi = 0.62//2HV for the Ar"^ laser (A = 488 nm) and 2wi = 1.34/72^^for the Nd:YAG laser (A = 1060 nm), where 2wi is given in |xm.

C. Depth of Focus The depth of focus can be specified by A/, which is the distance between the points at each side of the beam-waist image, for which wi = V2wi. At these points the irradiance of the beam falls to one-half of its value. With the use of Eq. (6) and the definition s' = A//2, the depth of focus is given by A / = ^ ,

(10)

which combined with Eq. (9), yields the expression

^f'2Si(4^f.

(11)

In practical applications Eq. (11) corresponds to

for the Ar"^ laser and A/=2.58(-^ for the Nd:YAG laser, with A/ given in |xm.

D. Irradiance at the Image Waist The irradiance at the image-beam waist is an important experimental factor, as high irradiance can damage samples. The fraction of the total power P of a Gaussian beam which is contained within a radial aperture r =wi is

Instrumentation

55

approximately equal to 86%. Thus, the mean value of the irradiance at the image-beam waist becomes equal to

m

0.68P/2W

(11.1)

E. Exact Lens Relation for a Gaussian Beam

The laser beam is often focused with a lens which is located in the near field of the incident beam. The resulting characteristics of the beam may be very different from those described by the classical analysis of geometrical optics. The standard lens formula is

1 1_i

(12)

s^ s' " / ' with s and s' the object and image distances, respectively, and / the focal length of the lens. For the case of a Gaussian beam (see Fig. 3) this relation becomes 1 5 + 2^/(5-/)

1

+ -T = S

(13)

/'

where s and s' are the waist-object and waist-image distances, respectively. The Rayleigh range was defined above by ZR = 7r(2wo)^/4A. The limit as ZR^-O corresponds to the geometrical case. If the incident-beam waist is located at the front focus, the emerging beam has a waist at the back focus.

ZR

OBJECT

Z-R. IMAGE

Figure 3 Imaging of a Gaussian beam by a positive lens.

56

M. Delhaye et al.

The maxima and minima of 5', which are of particular interest, can be obtained by differentiating Eq. (13). Thus,

and

fors=f(l±z^/f). As an example, consider the case of an Ar"^ laser beam (A = 488 nm) with a full divergence of 0.1 mrad focused with a lens of focal length 50 mm. If Z R » / , then 5min = '^max^/- F^r all object distances s the image waist is near the back focus. The magnification is then given by ^ =^

2wo

= T7v—J7^2-7-r^^ini2'

[{l-s/ff^{z^/fn

(16)

The minimum value of 2WQ is obtained for ^ » / and the size of the image waist is equal to 2w' ~/^o-

F. Laser Beam Quality

The modes employed in laser sources are not always Umited to the fundamental, TEMoo- The fundamental mode is often mixed with one or more higher ones. In order to specify the quality of a laser beam, a figure of merit Q has been defined by*

er=Qeo.

(17)

where 6^ is the real, far-field divergence of the beam. Thus, Eq. (5) becomes ^,= 1 . 2 7 : ^ ,

(18)

where Q= I for the TEMoo mode and Q > 1 if higher-order modes are involved. High-power gas lasers and solid-state lasers, such as the Nd:YAG, have Q values from 1 or 2, up to 200, depending on the technology. The value of Q allows the beam characteristics, and hence the beam focusing, to be specified. The real beam can be treated as Gaussian by substituting an *(2, as defined here, is the same as M^ used in the American Hterature.

Instrumentation

57

'artificial wavelength' QX into the equations employed above for a TEMQO beam. As an example, the beam diameter of a real, focused laser beam is given by

The irradiance at the beam waist is then equal to

It should be noted that in certain commercial instruments the figure of merit Q has been measured.

III. MICROSCOPE OBJECTIVES A. Characteristics

The microscope objective is undoubtedly the heart of the Raman microspectrometer, as it plays the most important role of coupUng both the light source and the spectral analyzer to the specimen. All of the potential information, spectral as well as spatial, is contained in the pupil of the objective and in the primary enlarged image of the sample. In principle, there are no fundamental differences between the optical systems used in conventional Raman instruments for macrosamples and the special optical devices devoted to microprobe analysis, since they both obey the same physical laws. However, micro-Raman systems impose more severe constraints in order to preserve a high spatial resolution and to maximize the signal, with the use of wide-aperture optical components. In particular, an exact matching of all intermediary pupils and images along the optical path is of primary importance, as indicated in Chapter 2. Unlike the classical 90° arrangement, which requires relatively long working distances with the use of two separate lenses, almost all microRaman instruments employ a single, wide-aperture objective in the 180°, or backscattering configuration. A beamspUtter is needed in order to illuminate the sample with the laser beam coaxially, through the objective, and to transmit the backscattered radiation toward the spectral analyzer. The use of commercially available microscope objectives has prevailed in most instruments, as these elements are almost perfectly corrected for aberrations over the relatively narrow spectral range employed in Raman spectroscopy (Wilson and Sheppard, 1984; Keller, 1989).

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Microscope objectives are essentially short-focus, large-aperture aplanetic systems. The cone of light focused or collected by the objective is determined by the numerical aperture (N.A.), which is defined by N.A. =Azsin^,

(21)

where n is the real refractive index of the medium between the object and the lens and B is the semi-angle of the cone for an axial objective point. Commercially available objectives exhibit a maximum semi-angle of ^ — 72°, or N.A. = 0.95 in air, where n = \. In order to introduce aberration corrections these objectives must be used under conditions specified by a fixed magnification factor y and a lens-toimage distance which is usually referred to as the 'tube length'. These specifications are always precisely given by the microscope maker and are sometimes engraved on the barrel of the objective. Consequently, the distance between the object and the front optical element, which is called the working distance (WD), as well as n, the index of refraction of the optical medium in the object space, must be strictly respected. The effective focal length/o is a constant characteristic of a given objective, which is often not specified. It can be deduced with the use of the classical lens equation from the value of 7, the magnification normalized at a fixed tube length p . The latter quantity may vary from 160 to 190 mm, depending on the maker's standards. Infinity-corrected objectives are often preferred for Raman experiments. These lenses are designed to form a parallel output beam when a point fight source is placed at the front focus. An additional convergent achromat, referred to as the 'tube lens', is needed to form a real image of the object. An advantage of this combination is that the effective magnification y^ can be varied without degrading the correction, by choosing the appropriate focal length /t of the tube lens achromat; thus, y^ =/t//o-

B. Efficiency of Light Collection The basic equations which describe the origin of the Raman signal intensity were presented in Chapter 2. The polarization properties of both isotropic and anisotropic media, e.g. oriented crystals, were considered. In practice, the lack of knowledge of the precise geometry of a specimen and the random orientation of |xm-sized particles or heterogeneous sample inclusions makes virtually impossible the prediction of the spatial distribution of the Raman scattering intensity. A limited number of experimental data have been reported which indicate that the 'scattering indicatrix', depicted as a three-dimensional plot of the observed intensity in all directions of space, may be significantly different from that predicted by the theoretical analysis.

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59

Figure 4 The centered spherical indicatrix. In order to provide the user with a practical basis for the comparison of different kinds of microscope objective, two simple geometrical models can be proposed. They are based on two different assumptions, namely: (i) a spherical indicatrix centered on a punctual sample, or (ii) Lambertian scattering for which the intensity vanishes in the plane of the specimen. 1. Isotropic Point Source In this model the sample is assumed to be a point source O which radiates uniformly in all space, i.e. fl = 47? steradians. For consistency with most applications in optics, the traditional calculation refers to the half space where n = 277, although it should be pointed out that with certain special optics a wider soUd angle can be covered. The emission indicatrix is a sphere whose center coincides with the source. Thus, the irradiance / = dc^/d^, expressed in W m~^, at the entrance aperture S of the light collector is constant for all directions in space. The total signal collected in the acceptance cone of the objective is obtained by integrating the Hght flux 0 over the solid angle ft (Fig. 4). For a limited, axially symmetric, conical beam of semi-aperture S the solid angle ft is given by ft = 277(1 - cos e) = 477 sin^ j ,

(22)

so that the light flux becomes <^t = 3277(1-cos ^), where 3 is the intensity of the light.

(23)

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M. Delhaye et a/.

Figure 5

The annular objective.

For an annular light collector such as the Cassegrain-Schwarzschild system, the solid angle for an axially symmetric spherical zone limited by the internal angle ^o ^^d the external angle 6i must be calculated. The result is shown in Fig. 5. Thus, ft = 27r(cos ^0 ~ cos ^i).

(24)

z <

5

< uu H

a O

z < Q

O in

0

0.2

0.4 •

0.6

0.8

1.0

NUMERICAL APERTURE Figure 6

Collection efficiency of a microscope objective.

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61

The curve given in Fig. 6 represents the dependence of the soHd angle ft and the Hght flux
Source

In the second, more traditional, model it is assumed that the sample is a flat, perfectly scattering surface S for which the available space is limited to ITT steradian. Lambert's law is based on the additional assumption that the brightness B is uniform in all directions. Thus, the intensity 3 exhibits a cosine dependence on the angle 6, d3 = 5 d 5 c o s ^ .

(25)

The three-dimensional indicatrix is then a sphere tangential to the sample plane at O (Fig. 7).

Figure 7 Lambertian scattering indicatrix.

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M. Delhaye et al.

100

0.2

0.4

0.6

0.8

1.0

NUMERICAL APERTURE N.A. = sin 0 Figures

Lambertian model ((/)ocsin^^).

With the use of this classical model, the collection efficiency is determined by integrating the light flux
TTB dS

sin 6 cos d dd

sin^ 6.

(26) (27)

The curve shown in Fig. 8 represents the dependence of the signal on the numerical aperture. To faciUtate comparison, the light flux is normalized to 100% for the half-space of 27r steradian. The hypothesis of Lambertian scattering leads to significant differences compared with the model based on uniform isotropic radiation. For example, typical values are 25% efficiency for N.A. = 0.5 and 8 1 % for N.A. = 0.9. This model emphasizes the importance of the paraxial rays and gives a decreasing role to the marginal ones. It must be pointed out that such simple models as those outhned above account only for the effect of the solid angle on the light-gathering power. Clearly, other factors, such as the quality of laser focusing, which determines the local irradiance, the matching to the other apertures in the coupling system, together with the optical attenuation over the whole path, can

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dramatically modify the overall response of a micro-Raman instrument. For these reasons, a vahd comparison of different objectives necessarily involves the measurement of signal intensity for the complete instrument, as will be illustrated in the following chapter.

C. Lens Objectives

7. Spherical Aberration Spherical aberration is the major cause of image degradation when an uncorrected, centered lens system is used to collect light from an axial point. When a wide-angle lens is employed to form a real image of the point, as shown in the example of Fig. 9, the marginal rays are focused at a shorter distance than are the paraxial rays. The rays issuing from the object point A with different angles di and O2 reach the lens surface at different radial distances hi and /z2, respectively. As the system is axially symmetric, the refracted rays incident at a constant angle d are convergent on a conjugate point A' on the axis. When the angle of incidence 0 and the radial distance h increase, the crossing-point A' is displaced along the axis to a shorter distance. The contour of the intersecting rays is a 'caustic surface' consisting of the total beam envelope. It is represented by a dotted line in Fig. 9, where the axial segment A^Ac is also shown; A ^ is the conjugate point for the marginal rays and Ac is the convergence point for the paraxial rays. The length of the axial segment A ^ Ac measures the longitudinal aberration, which is to a first approximation proportional to h^. Rather than a true point image, the uncorrected spherical lens produces on a viewing screen placed in the image region a 'blur' circle of radius p which is called the transverse aberration; it is proportional to h^. For moderate apertures a useful correction may be obtained by a proper choice of the curvature radii in a meniscus lens, or by combining positive and negative lenses.

CAUSTIC SURFACE A'

LONGITUDINAL ABERRATION

Figure 9 Spherical aberration of a wide-angle lens.

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M. Delhaye et a/.

A

1_«_. >^

\

O

\

«=1

fi

^

1—.-1^

C J

OPTICAL AXIS

C

J

^

Figure 10 The aplanetic points of a sphere of index of refraction n. The spherical aberration can also be described in terms of wavefront deformation. A plane wavefront perpendicular to the optical axis is not, in general, transformed into a spherical one. To compensate for this deformation, the lens can be designed with the use of aspherical surfaces of revolution (Meier et aL, 1972). Such components are marketed for special applications, such as compact-disc players, and benefit may be taken of their availabihty for Raman experiments. Another category of corrected lens, which is derived from fiber-optics technology, consists of a rod of graded optical index. A paraboUc radial distribution of the refractive index results in an optical cylinder with focusing properties analogous to those of a convergent objective. These optical components are marketed at moderate cost under the name SELFOC (1987). Special mention should be made of holographic lenses, which may in certain configurations play the role of high-aperture, convergent objectives. However, the presently available holographic objectives exhibit chromatic effects which may be troublesome under the conditions of most Raman experiments. The correction for spherical aberration in all modern high-aperture microscope lenses relies on the properties of a hemispherical-front lens of very short radius of curvature. It is employed under aplanetic conditions, as first proposed by Amici in 1830. As shown in Fig. 10, a sphere of radius R composed of an optical material of refractive index n placed in air, possesses two aplanetic points C and C. They are situated on the axis at distances Rn and R/n, respectively, from the center of curvature O. All rays crossing these points satisfy the Abbe sine condition. A point source placed at C yields a virtual image at C with a substantial magnification, y = n^, and without aberration, even at the maximum angle of refraction. The emergent cone of light has a reduced angular aperture, so that the complete compensation for spherical aberration, together with a balanced correction of the other aberrations, can be achieved. A series of menisci and compound lenses of different indices and dispersions is employed for this purpose (see Fig. 11).

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LOW APERTURE CORRECTION ELEMENTS

HIGH APERTURE HEMISPHERICAL FRONT LENS

APLANETIC COMPOUND LENS I FOCAL POINT

Figure 11 Cross-section of a typical high-aperture microscope objective. 2. Immersion

Objectives

The situation shown in Fig. 10 is encountered when a homogeneous immersion (HI) objective is employed. Figure 12a illustrates the case in which a half-sphere of optical glass is coupled to the specimen through a Hquid layer of the same refractive index. Often nonfluorescent cedar oil {n = 1.515) or synthetic organic mixtures are used. An advantage of the homogeneous immersion technique is a significant improvement in the numerical aperture, which is, for the same semiangular cone-angle d, muItipHed by the value of the index of refraction. The diffraction-limited resolution is thus significantly enhanced. Water immersion (WI) objectives in the visible region or glycerine immersion (GI) objectives in the UV region also merit particular attention. They have specific advantages, as in the case of water, whose index of refraction is n = 1.33; they significantly enhance the N.A. and, thus, the spatial resolution of the system. Water and aqueous solutions are poor Raman scatterers and are most often compatible with biological samples, as well as many samples of industrial interest. Furthermore, the immersion medium provides an efficient cooling effect which protects the specimen to some extent from overheating and subsequent thermal degradation by the laser excitation. Water immersion objectives with long working distances

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M. Delhaye et al. (c)

(b)

(a)

AIR IMMERSION OIL

I SAMPLE

SAMPLE

ni = 1 WATER OF MOUNTING MEDIUM n4

COVER GLASS OF THICKNESS e

"2

Figure 12 The different types of lens-microscope objective, (a) Homogeneous immersion, (b) 'Dry' metallurgical, (c) Biological, corrected for observation under a coverglass of specified index and thickness.

(WD) have recently been developed; for example, WD = 2 mm for y •• 40, a characteristic which can be useful in many applications. 3. Metallurgical and Biological Objectives In many experiments the interference of the Raman spectrum of the immersion liquid with weak bands of the specimen is not desirable; thus, many users prefer the 'dry', or metallurgical objectives. In this type of objective the sample is observed in air and a residual spherical aberration due to the frontal lens is compensated by corrective optical elements placed in the barrel (see Fig. 12b). In a similar manner the 'biological' microscope objectives are specially designed for the observation of specimens embedded or immersed in an aqueous medium through a cover glass of refractive index /I3 and thickness e, where typically n^ = 1.52 and e = 0.17 mm (see Fig. 12c). A limited range of adaptation to cover glasses of different thicknesses is provided by the manufacturers for certain objectives by adjustment of the internal spacing of the two groups of lenses, usually in the range 0.11-0.23 mm. Relatively weak spherical aberrations can be compensated in a first approximation by altering the effective tube length in order to introduce an aberration of opposite sign. However, as demonstrated by Sheppard and Min Gu (1991), the higher-order spherical aberrations produced by either a thick layer of a dielectric medium or those introduced by varying tube lengths do not result in complete cancellation. For practical reasons the modification

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of the tube length is not readily accessible to the user, as the position of the intermediary image is restricted by the optical configuration of the coupUng system (see Chapter 2). In the design of biological objectives an optimized correction for all aberrations has been made by a careful computation of the optical parameters of the whole set of compound lenses placed in the barrel. In particular, this overall correction takes into account the four refractive indices ^ i , /I2? ^3? ^4 and the exact thicknesses of the optical media which are present between the object and the lens (see Fig. 12c). Strange as it may seem, many users of Raman microprobes often ignore the very restrictive specifications imposed by the objective design. Thus, for example, samples are sometimes observed with a biological objective, but without the use of a coverglass. Such a misuse can result in serious performance degradation. Similarly, it is worth noting that the spatial resolution and throughput of a metallurgical 'dry' objective are adversely affected by the spherical aberration introduced by the plane diopter interface when an observation is made of an inclusion embedded in a high-index matrix. The effects of the uncorrected aberrations introduced by a plane diopter must not be overlooked. An estimate of their importance when a microsample is observed through a window of thickness e, or in a thick transparent matrix at a depth e, is shown in Fig. 13a. A simple ray tracing based on Snell's law shows that the apex of the caustic surface coincides with the position of the paraxial focus CQ when the objective is assumed to be focused onto a virtual point at depth ein. Obviously the marginal rays are focused much deeper, on C^. The longitudinal spherical aberration is defined as the distance

C ^ = ( ^ + 4)(^2^2)'y, \ ^2

(28)

^1/

where ni = l for air, ^2 is the refractive index of the matrix, 62 is the refraction angle of the marginal ray and y is the radial distance at which the marginal ray intercepts the interface. In some typical situations, e.g. inclusions in geological samples or specimens observed through a thick window in a cryostat, the longitudinal aberration may exceed 100 |xm. As a result, both the depth discrimination and the lateral resolution are severely impaired. In these cases confocal microscopy is not feasible. Accordingly, much care has to be taken in the design of special sampling devices so as to provide at least an approximate correction for the spherical aberration. This correction can be introduced by adding a compensating lens between the sample and the front lens of the objective. Such lenses are available from certain microscope manufacturers as accessories to be employed with objectives with a long working distance. The index of

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M. Delhaye et al.

OBJECTIVE FRONT LENS

CAUSTIC SURFACE

(b)

OBJECT PLANE

(c) OBJECTIVE FRONT LENS

OBJECT PLANE

Figure 13 (a) Aberrations introduced by a plane diopter, (b) and (c) Microsample observed through a window of thickness e.

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refraction of each lens, which is specified, is to be matched to the window or matrix material by means of an immersion Hquid. As discussed above, the principle of the optical correction depends on the aplanetic points of a sphere. It is thus not difficult to design a compensating lens for a particular experimental arrangement. The two possible lens configurations are shown in Figs 13b and 13c. The center of the sphere is itself an aplanetic point, since all rays passing through the center are normal to the surface and thus not refracted. This situation is depicted in Fig. 13b, where a special planoconvex lens of radius of curvature R has been introduced. It is fabricated from an optical glass of the same refractive index n2 as the matrix in which the sample is embedded. It is positioned so that the center of curvature O is coincident with the object. In principle, a thin layer of immersion hquid must be interposed to avoid refraction at the interface. The improvement in optical performance of this system over that of an uncorrected one is spectacular when a small field around O is observed. The other possible configuration is derived from the pair of aplanetic points at Rn and RIn, positioned in coincidence with the object and the virtual focal point of the objective, respectively (see Fig. 13c).

4. Other Aberrations A typical, modern high-aperture microscope objective is illustrated in Fig. 11. The hemispherical front lens is always associated with a complex assembly of lenses, fabricated from different optical materials. Sophisticated computer calculations and simulations enable the instrument maker to take advantage of a choice of radii of curvature, indices of refraction and dispersions to optimize the corrections for these aberrations, such as coma or chromatic variations of the magnification. The corrections can result in a balanced improvement in the field curvature and a reduction of astigmatism in off-axis regions. It should be noted that chromatic aberrations are usually not critical in traditional Raman appHcations, as the useful spectral region is relatively narrow. However, in resonance Raman spectroscopy the comparison of data obtained with the use of different excitation wavelengths, sometimes from the UV to the near-IR regions, has to be made with caution. It is particularly important to take into account the defocusing observed with lens objectives when the excitation is changed from the visible to the UV regions. This effect strongly decreases the irradiance and is associated with increasing absorption by the optical media. The result is a dramatic reduction in efficiency. Ordinary achromatic and apochromatic objectives differ essentially in the number of different optical materials used in their fabrication. Special new glasses enable the focal length or magnification to be equalized at more than

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two wavelengths, as well as to aid the flattening of the chromatic response over the visible spectral range. The optimal response of an apochromatic objective necessarily implies that it is used for visual or photographic imaging in conjunction with a dedicated compensation eyepiece, an element which is important in obtaining a balanced correction over a wide field of observation. In most micro-Raman systems the use of an eyepiece is prohibited for safety reasons. However, the coupling optical system employs the primary image provided by the objective alone. Fortunately, a narrow field of vision is sufficient for punctual analysis, so that complementary correction by the eyepiece is unnecessary.

D. Mirror Objectives Optical systems which employ only reflective surfaces have been proposed to overcome the Hmitations of lens objectives, namely chromatic aberrations and the light absorption of glass. The all-reflective microscope objective offers the obvious advantage of perfect achromaticity, which renders it of undisputed value for microspectrometry in the UV or IR spectral ranges. 1. Spherical Mirrors The most popular of the all-reflective configuration, which is often called the 'reverse Cassegrain' by analogy with the telescope, employs only two spherical reflecting surfaces. A particularly efficient system was described a century ago by Schwarzschild, who estabhshed the conditions necessary to form, with minimum aberration, an image at infinity with respect to a point source. This system is composed of two spherical mirrors, one concave (Mi) and the other convex (M2), of radii Ri and R2, respectively, which are positioned to be precisely concentric at point O (see Fig. 14). Three parameters define the focal length / and the frontal distance A, namely the radii of curvature Ri and R2 and the separation D between the mirrors. The system must fulfil the following, very simple conditions: / ? j = / ( V 5 + l) = 3.236/,

(29)

7^2 = / ( V 5 - l ) = 1.236/,

(30)

Z) = 2 /

(31)

^ = : / V 5 = 2.236/,

(32)

and

where the total length of the system is equal to A-\- D = 4.236/ As a result of the Schwarzschild calculation, three primary aberrations can be eliminated

Instrumentation

I ^2

71

I

F/gwr^ 74 The Cassegrain-Schwarzschild all-reflective objective.

to first order along the optical axis, viz. spherical aberration, coma and astigmatism. However, higher-order aberrations are not corrected. In addition to the field curvature, which is not a serious limitation in punctual microprobing, the main disadvantage of the Schwarzschild objective in Raman experiments is the central obscuration, given by p = r2lri = 0.447.

(33)

This effect severely impairs both the transmitted flux and the radial intensity distribution of the Gaussian laser beam (see Chapters 2 and 5). When the pupil is uniformly illuminated, the modulation transfer function (MTF) is typically half as great in the mid-spatial frequency response as that of a corrected lens objective with an equivalent N.A. However, at higher frequencies it is slightly better. The practical limit to the numerical aperture of commercially available reflective objectives appears to be N.A. = 0.50 for a focal length/of the order of 5 mm. In principle, higher values of the N.A. and shorter focal lengths are feasible at the expense of the field dimension and adjustment tolerances. Care must be exercised in positioning and centering the two elements. A comprehensive review of all-reflective systems has been published by Kingslake (1978). It may be useful to workers employing such configurations.

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2. Aspherical Mirrors With the use of computerized machines, modern optical technology offers the possibility of direct generation of on-axis and off-axis aspherical surfaces of revolution. A surface accuracy of A/10 or better can be obtained. Replication techniques are also widely used to manufacture a variety of aspherical mirrors at moderate cost. (a) Parabolic mirrors Concave parabolic surfaces produce a perfectly parallel light beam from a point source placed at the focus. Conversely, they focus an incident parallel beam to a perfectly corrected, diffraction-limited spot. Theoretically, all aberrations are zero on the axis, even for very large values of the N.A.; unfortunately, however, the field of view is also zero. In practice, the degradation of the wavefront remains acceptable within a narrow angular field, which is sufficient for punctual Raman microprobing. The profile of a parabolic mirror is given by the quadratic equation Y = X^/4f, where Y is the distance to a plane tangential to the vertex and perpendicular to the optical axis a n d / i s the axial focal length (see Fig. 15). The absence of spherical aberration results from a purely geometrical property of this profile, since a plane wave normal to the axis is transformed by reflection into a perfect spherical wave centered on the focus. Compared with a spherical mirror, the paraboloid exhibits a difference A called the 'aspherical departure', which increases as X"^, as given by the approximate formula A-;TP773-

256f'

(34)

A parabolic mirror is equivalent to a perfect achromatic objective corrected for infinity. It exhibits diffraction-limited focusing and imaging capability if the field is limited to a small zone around the focal point. The angular field of view 6 can be evaluated with the use of the relation e = 2,5\8\^], D

(35)

where S is the wavefront departure and D is the diameter of the mirror. (b) Elliptical mirrors By comparison with parabolic mirrors, concave ellipsoids can be used as microscope objectives which are perfectly corrected to form an image at a

Instrumentation

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X

Figure 15 Parabolic mirror objectives, (a) On-axis. (b) Off-axis.

fixed distance 2C from the point source at one of the two focal points (F^ or F2), as shown in Fig. 16. The profile of an elliptical mirror is given by the equation of its principal plane section, that is X^

•+

- 1 = 0,

(36)

with la = A A ' and b = V(«^ - c^), where 2c is the distance between the two focal points Fi and F2. Without entering into detailed ray-tracing calculations, a qualitative evaluation shows that the imaging capability appears to be quite acceptable for a centered, on-axis mirror with moderate angular aperture (Fig. 16a). However, the imaging is useless for large off-axis angles and high eccentricities, as shown in Fig. 16b. It was first pointed out by Rosasco (1980), that for a Raman microprobe configuration which employs an annular, ellipsoidal mirror objective, the total solid angle fix is given by 27r

ftT =

02

d(f)

sin 0dd = 27r(cos 6^ - cos 62).

(37)

01

An interesting characteristic of both eUiptical and parabolic objectives is that the solid angle of collection can surpass 277 steradian, as there is, in principle, no limitation to the maximum angular semi-aperture 6 for a point source placed at the focal point. Consequently, the definition of the numerical aperture N.A. = sin 6 is vaHd up to the value sin ^ = 1. However,

74

M. Delhaye et a I.

' X

Figure 16 Ellipsoidal mirrors, (a) Centered, on-axis. (b) Large off-axis condenser, (c) Annular.

the mirror can collect light from both sides of a plane section which contains the focal point and is normal to the principal axis. Clearly, the classical definition of the//number is not meaningful in this case - except for moderate apertures. In practice, the geometry of the sample or other optical components imposes limits on the maximum solid angle. However, the collection efficiency can be much greater than that of a lens objective. A special optical configuration was patented (CNRS-ANVAR, 1987) in which the solid angle was doubled to fl~47r steradian by coupling two

Instrumentation

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e, - 90° N.A.,-LO Figure 17

Double ellipsoidal mirror for light collection over nearly Air steradian.

OBJECTIVE N.A. = 0.80

Figure 18

OBJECT

OBJECTIVE N.A. - 0.80

Use of a beamsplitter to minimize light losses.

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M. Delhaye et a/.

COUPLING OBJECTIVE N.A. = 0.80 ELLIPSOIDAL MIRROR N.A. - 1.0

y//////M^///)mm ^>,

•

\

PLANE MIRROR

\ I ^ I

F2

M2

Figure 19 A possible simplification of the system shown in Fig. 18.

coaxial ellipsoidal mirrors, as shown in Fig. 17. In this configuration a point source placed at the common focal point Fi is imaged by each of the halves to the second focal points F2 and F2, which are coincident with the exit aperture at the vertex of the other half. The two output beams, in opposite directions along the principal axis, have to be coupled optically to the laser and the spectral analyzer by two external systems. An interesting application of this principle, which was proposed by DILOR (1987), is shown in Fig. 18. Both sides of a beamsplitter are employed to minimize the Ught losses by directing approximately half of the laser power to the ellipsoid on the left side and the other half to that on the right. The Raman-scattered Ught, which is collected at twice N.A. ~ 1, is transmitted to the spectrometer via the same beamsplitter. A possible simpHfication of this system can be achieved by replacing one of the half-ellipsoids by a plane mirror which supports a microsized sample near its center, as shown in Fig. 19 (CNRS-ANVAR, 1987). Such optical systems transform the cone of light collected over nearly 2TT steradian from the sample with N.A. ~ 1 into an exit beam of lower numerical aperture, e.g. N.A. = 0.8, which can be coupled easily to a conventional lens or mirror objective. The ellipsoidal mirror in this arrangement plays the role of the high-aperture front lens in a conventional microscope objective.

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E. Comparison of Microscope Objectives with Camera Lenses A wide variety of high-aperture lenses is commercially available for cinephotography and video cameras, which may be of interest for macro- or long-WD, micro-Raman experiments. In this case attention must be paid to certain important parameters in the selection of such lenses. Their design may be significantly different from that which is appropriate to Raman microscopy. Camera lenses are designed to balance all aberrations so as to cover the useful field, which is determined by the dimension of the photodetector. The dimension is typically from 24 mm X 36 mm down to approximately 8.5 mm X 8.5 mm. The spatial resolution is therefore adapted to the pixel dimension, typically from 50 to lOixm. The spatial resolution of camera lenses is usually given in line-pairs per mm (LPM), as evaluated qualitatively by observing the image of test patterns with the use of a square-wave profile. This profile consists of alternate transparent and opaque bars of equal width. A much more significant evaluation of the image quality is obtained by consideration of the modulation transfer function (MTF), which describes the ability of an optical system to transfer the object contrast to the image. The MTF is measured by forming the real image of a periodic test pattern which exhibits a sine-wave profile with variable spacing. The corresponding contrast or 'modulation' in the image plane is plotted in cycles per mm vs. the spatial frequency. It is worth noting that the art of camera lens fabrication reUes on an optimized balance of residual aberration in a wide-angle field at the expense of axial resolution. Typical resolutions range from 30 LPM for ordinary photographic cameras up to 200 or 500 LPM for photohthography. By comparison, the resolution necessary for micro-Raman measurements is of the order of 1000-2000 LPM. It is concluded, then, that camera lenses are satisfactory for macro- or semi-macro Raman spectroscopy with sample dimensions in the range 10-50 fxm, but are not suitable for diffraction-limited, laser focusing and Raman imaging. The value of the angular semi-aperture Q of camera lenses is generally not available in the manufacturer's specifications. However, it may be determined easily from the photographic //number, which is usually defined as the ratio of the focal length / to the pupil diameter D. Thus, in principle, //number = flD = 1/(2 tan 0).

(38)

Since the effective focal length has to be measured along the optical axis, this definition is not meaningful for very large values of 6, where tan 6 tends toward infinity. In practice, the //number is defined by 1/(2 sin ^), in agreement with Abbe's aplanetic condition. This relation is also in accord with photometric measurements on plane photodetectors, which obey

78

M . Delhaye et a/.

s

ASYMPTOTE

//o .

D H //2 CLH

5 S

//4 -

^5? ^

< ^ z^ o ^ H o

//6. //8-

C-

//lO

0.1

0.2

0.3

0.4

0.5

NUMERICAL APERTURE Figure 20 Relation between //number and numerical aperture.

Lambert's law. The //numbers are determined with the use of an iris diaphragm whose steps are arranged in a geometrical progression, i.e. //I

fllA

//2

//2.8

//4

...,

so as to double the light flux between two successive values. An approximate relation between the //number and the N.A. is given by //number — (2 N.A.)~^; thus the collection efficiency varies as l/(//number)^. To provide a convenient conversion, the graph given in Fig. 20 represents //number vs. N.A. in the range where camera lenses are available. An informative comparison with the collection efficiency of microscope objectives may be obtained by combining the diagram of Fig. 20 with those of Figs 6 and 8 (Section IILB. of this chapter). The practical limit of high-aperture camera lenses is //0.95. This value nearly corresponds to N.A. = 0.5, with a collection efficiency of miiT = 13.5% for the spherical indicatrix model and 25% for the Lambertian model. At//5 the value of N.A. becomes equal to 0.1 and the light-gathering efficiency falls to 1% or less.

F. Determination of the Pupil Dimension of a Microscope Objective The importance of the optical invariants which determine the throughput of the entire optical system of a Raman microscope will be emphasized in Section IV of this chapter. When the different elements of a micro-Raman

Instrumentation MICROSCOPE OBJECTIVE

79

SCALED RETICULE EYEPIECE

—--H-4-> *

/

1

LIGHT SOURCE

MEASURING EYE TELESCOPE DIFFUSING SCREEN Figure 21 Determination of pupil dimension and axial position (see text).

instrument are examined, it is found that the throughput is Hmited primarily by the circular pupil of the microscope lens and the entrance aperture or slit of the spectral analyzer. In general, the latter is not circularly symmetric. Among the parameters which are needed to calculate the throughput of a given microscope objective in a specific configuration, only the numerical aperture and the magnification factor are specified by the manufacturer. In order to complement these available data it is usually sufficient to measure the dimensions of the conjugate primary image, the effective tube length p (or the focal length/of the tube lens), and the pupil dimension in the image space of the microscope. The diameter and position of the objective pupil are parameters which are not readily available to the user. A direct measurement is often not feasible because the exit pupil can be virtual or inaccessible. The best solution to this problem is often to determine both the pupil dimension and the axial position by means of a small telescope equipped with a scaled reticule eyepiece, as shown in Fig. 21. Typical values of the diameter of the exit pupil are of the order of a few mm for high-aperture and high-magnification objectives, and up to 10 mm for low-magnification lenses.

IV. SPECTRAL ANALYZERS A. Introduction The polychromatic light beam collected by the objective of a Raman microprobe has to be analyzed by a spectrometric system. Thus, the spectrum - the detected light intensity as a function of the wavenumber - is recorded. The purpose of this section is to discuss briefly certain problems which arise from specific experimental situations encountered in Raman microscopy

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and the microprobe. No attempt will be made to analyze all possible spectrometers. In effect, all types of spectral analyzer and filtering system may be classified into two categories: dispersive analyzers and nondispersive analyzers.

1. Dispersive Analyzers A series of monochromatic images of the entrance aperture stop, usually a narrow slit, is spatially dispersed as a known function of wavenumber, with the use of prisms or diffraction gratings. These results are then displayed optically so as to form a real image of the spectrum. In a monochromator, an exit slit is positioned in the plane of the spectral image in order to isolate a narrow bandpass. The light flux passing through the slit is measured by a single photodetector during the sequential exploration of the spectrum. In a spectrograph, a portion of the output spectral image is directly focused onto an image detector, which usually consists of a Hnear array or a two-dimensional matrix of individual photodetectors. The spectrograph provides a simultaneous measurement of a large number of spectral elements.

2. Nondispersive

Analyzers

Various types of spectral analyzers and filters possess, as a common feature, the absence of a real spectral image. For instance, absorption or interference filters selectively transmit a part of the incident fight flux corresponding to a specific bandpass. An attractive approach is also offered by different types of interferometer, used in an optical configuration which do not require a narrow entrance sUt. The most popular instrument in this category, particularly in infrared spectroscopic applications, is the Michelson interferometer. In this instrument the different components of a polychromatic radiation are selectively modulated when the phase difference between the interfering beams is varied. The spectral data are decoded by means of a Fourier transform operation (see Section VIII of this chapter). Another type of system makes use of an encoding grid or mask placed in the plane of the spectral image. A selective modulation is produced by translating or rotating the encoding mask. The spectral information is decoded by a mathematical operation called the Hadamard transform, which is discussed briefly in Chapter 4 (Tilotta et aL, 1987; Tilotta and Fateley, 1988).

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2h

Figure 22 The geometrical extent of an optical system.

Before entering into a brief description of these different instrumental methods, it is essential to emphasize the features which are of prime importance in their particular application to Raman microscopy, namely (i) geometrical extent, (ii) instrumental spectral profile, and (iii) stray Hght background.

B. Geometrical Extent The ability of a spectral analyzer to maximize the light flux transferred from the source to the detector has been described by a variety of names, such as light-gathering power, light grasp, throughput or luminosity L. It is convenient to consider this quantity, say, the throughput, as a product of two factors; thus. L=

TU,

(39)

where T is the transmittance, taking into account all absorption, diffusion or reflection losses and U is the geometrical extent or 'etendue\ To define U, consider, as depicted in Fig. 22, a small object of area a perpendicular to the optical axis, at a distance / f r o m the entrance pupil of area ^ , in a medium of refractive index n. For a point O on the axis, the solid angle subtended by the entrance pupil is fl and the half-angle of acceptance is 6.

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Figure 23 The Helmholtz-Lagrange invariant. The geometrical extent, which is then given by U = n^asin^S 1 + cos ^ = n^an277

(40) (41)

is an invariant. It characterizes the optical system along the entire light path. In the small-angle approximation, and assuming AI = 1, as in air, Eq. (41) is simplified to aA

u = an =

T

(42)

Another invariant is sometimes introduced by considering a one-dimensional geometrical extent. It is often referred to as the Helmholtz-Lagrange invariant, given by h,dr = h2e2 = h^e3,

(43)

where h is the height of the object and the subscripts refer to the conjugated images (see Fig. 23). By substituting sin^ for the angle 6, Eq. (43) then corresponds to the Abbe sine condition, hsm6 = constant.

(44)

When the optical system is not circularly symmetric, as for instance in a slit monochromator, two values of the one-dimensional extent must be considered. A value is needed for each of the two orthogonal planes containing the optical axis, one parallel to the slit height and the other perpendicular to it.

C. Instrumental Spectral Profile and Bandpass By definition, the bandpass of a monochromator, or more generally of a spectral filtering device, is the spectral interval Av defined by two wavenumber limits vi and 1^2 when the system is illuminated by white light or a

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83

X D J u. H

Av

oa:

- •

-<

-I

V,

V2

^'l

WAVE]^[UMl3ER (cm ^)

(a)

WAVENUMBER (cm ')

(b)

FWHM

(c)

WAVENUMBER (cm'')

FWHM

(d)

WAVENUMBER (cm"^)

Figure 24 (a) Bandpass of an ideal spectral filter illuminated by white light, (b) The ideal spectrum of a monochromatic light source, (c) Instrumental profile, (d) Line profile sampling by the pixels of a multichannel detector.

continuous light source (see Fig. 24a). However, it is more informative to consider the spectral response of the system when it is illuminated by perfectly monochromatic radiation (Fig. 24b). The resulting instrumental profile, as shown in Fig. 24c, is determined by plotting the output flux (f)^ against the wavenumber D. In the ideal case the line width would be equal to zero. However, in reality an apparent spectral broadening occurs. The bandpass A? is traditionally defined as the full width at half maximum (FWHM), which in this case is due to the instrumental profile. Additional features of great interest are the attenuation slope on each side of the central-peak maximum, the existence of secondary peaks, or an asymmetry of the bandshape which can reveal mismatching of the slit to the line curvature, residual aberrations such as coma, or nonuniform illumination of the pupils.

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The resolvance power Dl is defined by the ratio of the absolute wavenumber to the FWHM, AV

FWHM •

^ ^

In the particular case of a mutichannel spectrograph, which has proven to be the most efficient spectral analyzer for Raman microprobes, the effect of discrete sampling of the instrumental profile by the multichannel photodetector has to be considered. Multichannel arrays consist of rectangular, slit-shaped elements, while the matrix of the charge-coupled device (CCD) type is made of square pixels whose dimensions range from 9 to 25 |xm (see Section V). A representative figure of merit is then the number of pixels for a spectral bandpass. It is usually assumed that a minimum of 3 to 7 pixels is necessary for a correct sampling of the band profile and a determination of the peak intensity (see Fig. 24d). As proposed independently by several authors, the sampUng of the profile may be improved by digital treatment of a series of data recorded by sUghtly shifting the spectral images with respect to the detector array between successive integration steps (Singer et al., 1988; DILOR, 1990; Hadbawnik and Kraiczek, 1990). Such treatments merit attention with regard to profile smoothing and may also be useful to modify the spectral range covered by a detector array. However, the FWHM cannot be defined by the dimension of a single pixel, since the sets of sequentially recorded data are not totally independent, but remain spatially correlated due to the pixel structure.

D. Stray Light Level

A number of situations arise in Raman microprobing whereby the detection of weak bands depends on the ability of the whole spectral analyzer system to reject efficiently the exciting laser radiation. The exciting radiation is scattered elastically by the sample medium with a much stronger intensity than the Raman scattered light. It is clear, then, that the backscattering configuration is more effective for the collection of this undesirable laser Ught than the traditional 90° configuration (see Chapter 2, Section IV). One direct consequence of the wide soHd angle of acceptance of microscope objectives is the extremely intense light flux at the laser wavelength. This fight can be reflected towards the spectrometer, via diffuse reflection, or even specular reflection, at the interfaces in the vicinity of the specimen. In the very particular situation of interest for Raman microprobing, stray Ught at the laser wavelength is largely preponderant. Under this restrictive condition, the estimation of the stray light rejection of a given system may be simpUfied by considering its instrumental profile under

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monochromatic illumination and measuring the background level
for an uncoated surface. Assuming a refractive index of n = 1.5 for the coverglass yields a reflection of 4%, i.e. 400 |xW for a laser power of 10 mW. The situation is even worse when contaminants are investigated at the surface of a high-index material, for instance a siHcon wafer which reflects approximately 30% of the incident power. In typical experimental situations, the laser power reflected onto the aperture stop of the instrument can be of the order of mW, even when the excitation power is kept at moderate values. Several techniques are available for alleviating the problem arising from specular reflectance, for example the use of antireflection-coated coverglasses. Also, as the laser beam is linearly polarized, the reflected Hght can be significantly attenuated by placing a crossed polarizer in the coupling optics. This procedure, however, results in a strong decrease in the intensity of the polarized Raman bands. Another technique introduced in the nineteenth century for metallography employs a set of two complementary screens each covering half of the pupil - in the excitation and coflection beams, respectively. The obvious drawbacks of this system are the loss of signal intensity, together with a degradation of the spatial resolution. On the other hand when, for example, the sample consists of a white, powdered soUd such as MgO or Ti02, there is no way to get rid of the diffuse reflectance of the laser light. In such a case 10-100 )JLW of power may be collected in the acceptance cone of the microscope objective. To illustrate how difficult it is to detect the weak Raman signal, it is helpful to convert the reflected laser power P into a number of photons per second Np as given by iVp = 5 X lO^^PA,

(47)

where A is the wavelength in nm. One mW of green Hght represents a flux of 2.5 X 10^^ photons per second. The backscattered light flux is then superimposed at the entrance of the spectral analyzer on that of the weak Raman signal, which typically ranges from 10 to 10^ photons per second. Thus the optical system of the instrument should ideally exhibit image formation

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M. Delhaye et a/.

capabilities with a dynamic range or contrast ratio from 10^ to 10^"^. Such extreme requirements are not obtainable with ordinary optical systems, and can only be approached by a cascade of several stages of spectral filtering devices. The latter must be carefully designed and coupled so as to reduce stray light to a minimum. The basic assumption is that the optical S/N of a combination of several spectral filtering systems placed in tandem will be the product of their individual S/N ratios. For a comparison of the different systems proposed to improve the stray fight rejection, it is worth remarking that the scattered fight flux which is superimposed at any wavenumber on the Raman signal varies in direct proportion to the total area of all of the aperture stops along the light path. Thus, any attempt to improve the throughput of a system may adversely affect the contrast figure. Finally, the background level (^b is proportional to the solid angle over which the detector pixel perceives the light due to all of the scattering media along the illuminated path. The above analysis would appear to be valid for the random scattering processes occurring in the optical components: lenses, mirrors, gratings, etc. Nevertheless, the various other origins of stray light must be carefully considered. Some experimental observations, such as the variation of the background curve, may suggest other interpretations. For instance, spurious reflections at optical surfaces, or from baffles or mechanical parts, rediffraction by the dispersing element of beams emergent at different wavenumbers, and/or backreflection of the output beams onto the photodetector, can produce an increase in the background level in certain spectral regions. The first method proposed to resolve these problems involved two or more identical spectral systems, possessing similar bandpass, coupled in series. In the design of dispersive spectrometers, double or triple monochromators were the preferred configuration in the first generation of Raman microprobes. Assuming that the contrast ratio of a single grating stage is of the order of 10"^, a triple system could in principle attain 10^^. A valuable advantage of such instruments is the significant improvement of the instrumental spectral profile, which exhibits a triangular shape with a rapid cutoff on each side of the central peak. More recently, a different approach has consisted of coupfing two separate spectral filtering sections which possess entirely different profiles, e.g. a prefilter, or fore-monochromator, placed between the microscope output and the entrance aperture stop of the main spectral analyzer. The first stage has to be specially designed to produce a strong rejection at the laser wavelength, together with a broad transmission band covering the Raman spectral region (see Fig. 24a). A main spectral analyzer, most often placed second, can be dispersive or nondispersive. As it does not receive the intense laser radiation, which is suppressed by the prefilter, the essential function of this part of the instrument is to obtain the narrow bandpass needed to attain the necessary

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spectral resolution. The obvious advantage of this concept relies on a complete separation of the roles played by the two successive stages, which can therefore be optimized independently. In particular, the prefilter stage can be adjusted to yield its best rejection efficiency at a fixed wavelength of a given laser excitation. Finally, one of the most valuable advantages of the confocal microscope system is its ability to reject by spatial filtering the light at any wavenumber, including the laser radiation which originates from the out-of-focus regions. In a sense, the pinhole spatial filter effectively contributes to the reduction of the intensity of undesirable fight which enters the spectrometer. It should also be pointed out that diffuse scattering, fluorescence, or Raman scattering occurring in the optical media often produce a significant contribution to the observed spectral background. These contributions arise along the light path which is common to the excitation and collection beams, from the sample to the beamsplitter. For instance, the pure rotation and vibration-rotation spectra of oxygen and nitrogen, as well as the spectra of the optical glasses in the system, are easily observable with the use of a Raman microprobe in the absence of a sample. Such spurious spectra may also interfere with the observation of a sample spectrum and create artefacts. Efficient spatial filtering in a confocal configuration can help the experimentalist when weak Raman bands are expected in the spectral range where these interferences appear.

E. Dispersive Monochromators A monochromator can be represented schematically as in Fig. 25. A dispersive element is illuminated by a parallel fight beam which is produced by a collimator consisting of an entrance slit of width s^ and height hi placed at the focus of a convergent lens or mirror of focal length / i . A second collimator collects the fight beam diffracted at a given angle and focuses it onto the exit slit. Traditionally the dispersive element is a plane grating, which offers better efficiency and dispersion than a prism. Holographic gratings are preferred to ruled gratings owing to their advantage in eliminating ghosts and their extremely low stray light level. Recently the use of volume-phase holograms as transmission gratings has been reported (Carrabba et aL, 1990; Battey et al, 1993). Instead of a plane grating, a concave holographic grating can be employed. In this case the grating itself focuses the divergent beam originating from the entrance sfit onto the exit sUt (Fig. 26). Extensive studies of the properties of plane and concave gratings may be found in the fiterature and in the documents provided by the instrument makers (Instruments, S.A., 1979; Woods et al., 1994). The resolvance of a grating monochromator cannot exceed a limiting value

88

M. Delhaye et a/. PLANE GRATING COLLIMATOR

Figure 25

The basic schematic diagram of a monochromator.

CONCAVE HOLOGRAPHIC GRATING

ENTRANCE SLIT \

EXIT SLIT

Figure 26

Principle of a concave holographic monochromator.

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fixed by diffraction. It is called the intrinsic resolvance %) of the grating, which is given by 9^ = / v ^ ( ^ ) ,

(48)

where / is the width of the beam at the output of the disperser and {ddldv) is the angular dispersion. A well-known expression indicates that the maximum resolvance of a grating is proportional to the total number of grooves N^ and to the diffraction order k\ thus, %) = NA' (49) For example, in a double-additive monochromator composed of two gratings of width 100 mm, with 1000 fines per mm in the first order, the intrinsic resolvance 9to = 200 000. Thus, the lowest spectral bandpass 1^V = ^QIV would be limited for infinitely narrow slits to 0.1 cm~^ at A = 500nm (i^ = 20 000cm-^). In practice, one has to consider the angular width a and the angular height j8 of the sfits, assumed to be equal for entrance and exit slits, thus •^1

^2

(50)

and

^ 4 = ^.

(51)

The spectral bandwidth AT' is proportional to the angular width a, M) = — - —

(52)

and the resolvance, which is inversely proportional to a, then becomes equal to a\av J The geometrical extent U [see Eq. (40)] is given by U = Aap = A ^ ,

(54)

/i

where A is the cross-section of the beam, i.e. the projection of the grating onto the cofiimator. The throughput L, which determines the fight flux received by the photodetector coupled to the exit slit, is equal to L = TAal3,

(55)

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M. Delhaye et a/.

It should be noted that the product throughput-times-resolvance is entirely determined by the initial choice of the optical components, namely m=TApvl^\.

(56)

This product is clearly proportional to the angular dispersion of the grating, (d^/di^), and to the cross-section A of the beam. In practice, the resolvance is much lower than its intrinsic value. Furthermore, the product LJR is independent of the sUtwidth, as well as the apertures (or //numbers) of the entrance and exit collimators. This result explains why relatively long focal lengths and low-aperture spherical mirrors are most often employed. Thus, the aberrations can be kept to an acceptable level. A reasonable amount of astigmatism due to off-axis mirrors can be tolerated if the slits are parallel to the tangential focus. Similar considerations apply to concave holographic gratings, for which the astigmatism can be corrected within a narrow spectral domain.

F. Dispersive Spectrographs With the use of the same kind of entrance coUimator and disperser as that employed in the monochromator, the spectrograph is characterized by the properties of the exit section. This element projects the spectrum onto the multichannel detector, as shown in Fig. 27a. The function of the objective Lj of the spectrograph is comparable to that of a photographic or video camera. The throughput is determined by evaluation of the illuminance of a pixel in the detector plane. This quantity is to a first approximation proportional to the solid angle ftg subtended at this pixel by the cross-section of the beam at the exit aperture A^. The luminosity L of a spectrograph [Eq. (39)] is then essentially determined by the square of the numerical aperture of the objective, L2; thus, L=m^

TA

= ^ .

(57)

f: In practical applications the resolvance power is determined by the ratio of the objective focal length/2 to the dimension b of the detector pixel. Note that the objective has to be corrected for aberrations over the whole field, as determined by the dimensions of the multichannel detector. From this point of view, it appears that off-axis spherical mirrors, which are widely used as entrance coUimators, are not suitable for that purpose when high-aperture output objectives are employed.

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It may be recalled that three principal aberrations limit the imaging capabilities of spherical mirrors, namely: (i) spherical aberration (ii) astigmatism (iii) coma. The longitudinal spherical aberration / is defined as the difference between the focal distances of the paraxial and marginal rays (see Fig. 28). Then, 1=^ =^ , (58) 4 32/' ^ ' where r is the radius of curvature, w is the half-angle subtended by the mirror at the center of curvature C, and D is the diameter of the mirror. For an incident parallel beam, the spot size is determined by ^=—

= 64^'

^''^

where t is the transverse spherical aberration, i.e. the radius of the spot. For a half-mirror of width A = DI2 in the off-axis configuration, the longitudinal aberration remains unchanged, while the transverse aberration becomes equal to tl2. If the astigmatism produced by an off-axis spherical reflector is considered, it is found that a parallel beam is not convergent on a single point, but forms its best foci on two perpendicular straight lines, called the tangential focus and radial focus. They are separated by a distance a, which is given by a=fsm^y,

(60)

as illustrated in Fig. 29. In the case of spectrograph objectives, the angle y varies with the dispersion, so that the astigmatism is not constant over the detector area. The analysis of coma aberration is much more complicated and beyond the scope of this chapter. It should, however, be recalled that coma introduces an unacceptable asymmetry of the lineshapes which rapidly increases with the aperture and off-axis angle of the mirror. Considering the overall effects of these aberrations, it becomes apparent that a practical Umit to the aperture of the spherical mirror used at the output of a multichannel spectrograph is of the order of//8. For higher apertures, which are needed to optimize the irradiance over the detector pixels, compound lens objectives will perform much better than spherical mirrors, since a number of optical parameters, e.g. curvature indices and dispersion, can be exploited by the lens makers to minimize the aberrations over a wide field. Well-corrected lens objectives are available up to//1.5, a range which adequately covers the requirements of CCD photodetectors in the visible and

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LONGITUDINAL SPHERICAL ABERRATION

Figure 28 A spherical mirror receiving a parallel beam focuses the marginal rays m at a shorter distance than the paraxial rays p.

RADL\L OR SAGITTAL FOCUS

Figure 29 Astigmatism of an off-axis spherical mirror. near-IR spectral regions. Unfortunately the extension to the UV is not straightforward, since lens objectives are not currently marketed for this spectral domain. The same remarks apply to imaging spectrographs, in which a set of spectra originating from different points of a heterogeneous sample focused along the entrance slit are displayed on a two-dimensional multichannel photodetector, as shown in Fig. 27b. The spatial resolution which exists in the primary image in the plane of the entrance slit must be preserved and

Instrumentation

95

transferred without alteration onto a row of pixels of the photodetector matrix. A careful astigmatic correction is therefore of particular importance in imaging spectrographs (see Chapter 4). Hence, lens objectives are the most suitable for such instruments.

G. The Fabry-Perot Interferometer The Fabry-Perot system is a multiwave interferometer consisting of two semi-reflective surfaces Mi and M2 accurately positioned to be exactly parallel at a distance e, as illustrated in Fig. 30. For most spectroscopic applications, the mirrors Mi and M2 are planar, although spherical interferometers are widely employed in laser resonant cavities. When illuminated by an extended light source, the system produces circular interference fringes located at infinity, which can be displayed as a real image in the focal plane of a convergent element - mirror or lens, L2. The transmission function T of the system is given by the relationship T =

1 + m siv^ilirvne cos a)'

(61)

where a is an angle of incidence on mirrors Mi and M2, and T^ and m depend on the characteristics of the reflective surfaces. A series of transmission maxima, corresponding to the successive integer values of the order of interference /?, are observed when the phase difference {lirvne Qos a) satisfies the relationship sin^(7rp) = ^ix^ilTTvne cos a) = 0.

(62)

Figure 30 Principle of the Fabry-Perot interferometer. The curve depicted on the right shows the radial intensity distribution in the circular fringes, as produced by an extended monochromatic source.

96

M. Delhaye et a/.

R = 0.005

PHASE DIFFERENCE, 5 Figure 31 Variation of the transmission of a Fabry-Perot etalon for various values of the reflectivity R of the mirrors.

Note that the Fabry-Perot interferometer is basically a dispersive system, as the radius of the circular fringes varies as a function of the wavenumber v of the incident radiation. Usually, however, the central fringe is isolated from the ring pattern by means of a circular diaphragm O2, optically conjugated with an entrance aperture Oi by the two lenses Li and L2. Then the system is axially symmetric and performs as a nondispersive spectral filter. In this form, the interferometer is called a Fabry-Perot etalon. Its spectral properties can be modified by changing either the refractive index n of the medium or the mirror spacing e. Interferential filters employ the same optical configuration with the use of multilayered reflective surfaces in an all solid-state device. The instrumental profile of a Fabry-Perot interferometer is given by an Airy function, as depicted in Fig. 31. The free spectral range (FSR), which is the interval between two adjacent maxima, is given in cm~^ by FSR =

_1_ 2ne'

(63)

The spectral bandwidth of an Airy peak, defined by the FWHM, is equal to FWHM =

1 2neF '

(64)

Instrumentation

97

where F is known as the 'finesse'. Assuming that Mi and M2 are perfectly plane mirrors of reflectance R, the finesse is expressed by

For example, with /? = 0.9 the finesse is in principle equal to 29.7. However, any departure from the ideal mirror quality due to imperfections in the optical surfaces modifies the value of the finesse and reduces the spectral resolution. The defect finesse produced by a wavefront error AW is given by Fd = Nil. The contrast, defined as the ratio of maximum to minimum transmittance in the Airy profile, is given by ^min

\\-R

For the same example, with R = 0.9 the contrast reaches a value of 360. The intrinsic resolvance KQ is equal to the finesse times the order of interference p\ thus,

%. = i^ = pF-

(67)

As/7 is theoretically unlimited, the Fabry-Perot interferometer is an excellent system for obtaining high spectral resolution, at the expense of a very narrow FSR. The geometrical extent U of the interferometer exhibits a dramatic improvement by comparison with a grating monochromator of the same spectral resolution, because the narrow entrance slit is replaced by a circular aperture stop of much larger area. This property certainly constitutes the most important advantage of the Fabry-Perot system.

H. The Michelson Interferometer

Fourier transform (FT) interferometry is now a well-established technique which has proven of undisputed value in infrared spectroscopy. It is not the purpose of this chapter to describe in detail all of the features of FT instruments, which can be found in a number of excellent books and articles (ASPEN, 1970; Chase, 1987; Brenan and Hunter, 1994). It is sufficient to emphasize some of the basic properties of the Michelson interferometer in order to compare it with other spectral analyzers, as they are employed in micro-Raman experiments.

98

M. Delhaye et a/.

Mo

Figure 32 Optical configuration of a Michelson interferometer. BS, beamsplitter.

The basic optical configuration of the Michelson interferometer is shown in Fig. 32. The light beam which enters aperture stop Oi is collimated by a lens or mirror Lj and separated by a beamsplitter into two beams, in principle of equal intensity. After reflection by the plane mirrors Mi and M2, these beams are recombined to interfere. An exit collimator L2 focuses the interference fringe pattern onto a diaphragm O2, which is coupled to a single-channel photodetector. The light path difference 8 = Ine between the two interfering waves can be varied by moving the mirror M2, while keeping it parallel to the image MJ of the fixed mirror Mi. Assuming an incident monochromatic Hght beam of wavenumber v and a linear displacement of M2 at a constant speed V, it can be shown that the output light flux exhibits a sinusoidal intensity modulation at a frequency which is proportional to both v and V. A similar analysis for the case of a polychromatic source yields a complex interferogram which corresponds to the superposition of all of the modulated signals originating from different spectral components. This interferogram, which is a function of 5, is proportional to the real part of the Fourier

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Figure 33 Comparison of the spectral profiles of (a) two-wave and (b) multiple-wave interferometers.

transform of the spectral density of the incident light flux. It can be decoded by the inverse Fourier transform to recover the spectrum (see Section VIII of this chapter). This method has made remarkable advances since the introduction of mini- and micro-computers, which offer the capability of rapid Fourier transformation of a large number of points, together with powerful data treatment and many instrumental automation applications. To a certain extent, the optical properties of a Michelson interferometer can be compared to those of the Fabry-Perot configuration The essential difference lies in the principle of operation, which in the former case involves two waves rather than multiple ones. Thus, in the former instrument the response to a monochromatic radiation I{S) is sinusoidal. As shown in Fig. 33, the Airy function of a Fabry-Perot interferometer benefits from the multiwave interference which produces a significant narrowing of the transmission peaks. In the Michelson interferometer the spectral resolution is determined essentially by the maximum displacement of the mirror M2. This condition imposes an upper limit to the light path difference 3^. The half-width of the instrument profile for a monochromatic radiation is then given by ^Vy2

••

(68)

and the resolvance becomes equal to KO=VSM.

(69)

In order to reduce the amplitude of spurious secondary maxima (the 'Gibb's ears'), the interferogram is usually weighted by an apodization function, at the expense of spectral resolution.

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The two main advantages of the FT method can be briefly summarized as follows: (i) The multiplex or Felgett advantage, which is due to the modulation of all of the spectral elements, received simultaneously by a single photodetector. For A^ resolution elements in the observed spectral range, the signal-tonoise ratio increases as VA^. However, when different origins of noise in the photodetector are considered, it should be pointed out that the Felgett advantage applies only when the noise is independent of the signal level. This situation is effectively the case in IR absorption spectrometry. However, the extension to Raman microspectrometry is not straightforward, since modern high-performance photodetectors in the visible or near-IR (NIR) regions may approach the shot-noise-limited regime, in which the noise increases as the square root of the total light flux entering the photodetector, as shown in the following section (Girard, 1984). (ii) The Jacquinot advantage ('etendue gain'), due to the much greater throughput, can be directly compared to the equivalent feature of the Fabry-Perot interferometer. The geometrical extent of a Michelson instrument is determined by the area of the entrance aperture stop Oi, by the cross-section of the beams at M^ and M2, and by the focal length of the collimator Li. It should be recalled that the resolvance JK of the interferometer is related to the angular aperture 2a = d/f of the entrance diaphragm Oi, as given by the equation derived by Jacquinot, namely 1 AV lldV where d is the diameter of the aperture stop Oi and / i s the focal length of Li. As an example, a spectral bandwidth of Av = lcm~^ at lOOOOcm"^ (A = 1 |xm) corresponds to 31 = 10 000 and requires an angular aperture of ^<28xl0-^

(71)

This condition corresponds to a maximum diaphragm diameter of d = 5.6 mm with the use of a collimator of focal length / = 200 mm. Consequently, the divergence of the entrance and output beams must not exceed the angle required for the desired spectral resolution. Fortunately, this condition is not restrictive in most micro-Raman analytical studies. I. Prefiltering Devices

As illustrated in Fig. 34a, the spectral response of a prefilter stage would ideally exhibit a rectangular form, often called a 'notch', with effectively zero

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WAVENUMBER (cm ')

Figure 34 (a) The spectral response of an ideal notch filter, (b) A possible prefilter response for analytical measurements, excluding the anti-Stokes spectrum.

transmission in a narrow spectral domain centred on a given wavelength, e.g. the laser wavelength AQ. When selecting a prefilter or a fore-monochromator, the features of importance are the following: (i) High contrast, with minimum residual transmittance at AQ. (ii) Sharp cutoff and steep attenuation slope, so as to avoid the loss of intensity measured in the low-frequency Raman spectra.

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(iii) Flat maximum response, with high transmittance over the Raman spectral range. Typically this range covers 3500 cm~^ in the Stokes region and 1000 cm~^ in the anti-Stokes Raman spectrum. The latter is often not exploited. In this case a step spectral response, obtained with what is often called an 'edge filter', is suitable for analytical applications (Fig. 34b). 1. Gratings The most widely used optical prefiltering system consists of a double subtractive grating monochromator in a symmetrical optical configuration. Thus, the radiation dispersed by the first grating is recombined by the second grating. A real image of the dispersed spectrum is formed in the intermediate plane of symmetry. The slope of the spectral response and the cutoff can be easily adjusted by placing in this plane an opaque mask provided with a rectangular aperture, or a variable-width slit. Owing to the excellent stray light attributes of modern holographic gratings, the system may exhibit a very high contrast and an efficient light-rejection capability. By choosing the appropriate grating dispersion, the effective cutoff can be very steep and can be adjusted close to the exciting radiation. Access to the low-frequency Raman features, down to less than 1 0 c m ~ \ can then be achieved. The main advantage of grating fore-monochromators is their flexibility, provided for instance by computer control of the angular position of the grating, which allows the system to be tunable over a wide spectral domain. Hence, various laser excitations can be accommodated. The throughput limitation has been often presented as a drawback of this prefiltering system. With the use of two gratings in tandem, the transmittance typically reaches 0.5. The geometrical extent is essentially limited by the sht aperture. However, this system is well adapted to match the coupling to a microscope and to a dispersive monochromator or spectrograph. In contrast, a grating fore-monochromator is, in principle, not suitable to illuminate correctly the wide entrance aperture of an interferometer. Such a mismatched coupling would greatly diminish the Jacquinot advantage. 2. Interference Filters Interference filters are based on the principle of the Fabry-Perot interferometer. In this case the resonant cavity consists of a thin layer of soUd dielectric of thickness A/2 separating two reflective layers fabricated with the use of optical thin-film techniques. Multilayered, dielectric-reflector stacks are employed to increase the reflectance and thus decrease the bandwidth. Several Fabry-Perot elements can be combined in a multicavity filter to

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obtain the desired bandwidth with an improved rejection of the unwanted wavelengths. An additional blocking by absorption filters is often required. The performance of interference filters is specified for a well-coUimated beam normal to the surface. Attention must be paid to the dependence of the bandwidth and central wavelength on the angle of incidence a, as Amax = Ao[l - {n^ln.f

sin^ af'^,

(72)

where «o = 1 in air. In Eq. (72), n^ is the effective refractive index of the spacer medium, which is different from that of the bulk material. The user should be aware of the effects of changing the angle of incidence or using a divergent or convergent beam, which always decrease the transmittance. Furthermore, the bandpass profile is broadened and becomes asymmetric. In the absence of additional blocking filters, the resonant cavity can be used in either transmission or reflection. The spectral responses of these two arrangements are complementary, as shown in Fig. 35. This feature provides a means of rejecting the laser Hght in a Raman microprobe with the use of sequential reflections on several narrow-band interference filters (Puppels et al., 1991). Such sets of multiple interference filters may offer a much better efficiency than grating monochromators. However, the access to the lowwavenumber range is severely limited, and the spectral response is not flat over the usual Raman domain.

J. Bragg-diffraction Filters An entirely new type of filter has been developed by Asher et al. (1986). It is an efficient rejection filter consisting of a three-dimensional, ordered array of charged polymer microspheres in colloidal suspension. By adjusting the concentration, the pseudo-lattice parameter is made comparable to the wavelength of visible light. The filters are prepared by orienting the lattice between a pair of quartz plates. The diffraction of light on the filter produces a selective reflection for a wavelength AQ which satisfies Braggs equation, mAo = 2nd sin 6,

(73)

where 0 is the complement of the incidence angle, m is the order of diffraction, n is the refractive index of the suspension medium and d is the distance between lattice planes. When the incident light beam is polychromatic all wavelengths that do not fulfill the Bragg condition are transmitted, while a sharp spectral band centered on AQ is rejected. This filter thus displays an exceflent notch response with a bandwidth of the order of 10 nm and a residual transmission at AQ of <10~'*. The rejection wavelength can be adjusted in coincidence with the laser line by tilting the filter. In spite of its attractive features, this type of

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:z;

o H

u w

WAVENUMBER cm"

O C/5

^

WAVENUMBER cm~

Figure 35 The transmission and reflection spectral responses of a Fabry-Perot resonant cavity. filter is not often used, as the colloidal suspensions are not stable for long periods of time. 4, Holographic Phase-grating Filters A similar Bragg diffraction phenomenon can be observed with volume gratings. These elements consist of a transparent material in which the refractive index is spatially modulated (Solymar and Cooke, 1981; Pallister et al., 1992; Kim et al., 1993). The index modulation is most often produced to form parallel equidistant layers in which the modulation profile may be sinusoidal. The principle of this method was introduced more than a century ago by

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RAMAN ANTI-STOKES

Q

/

--

I

^, '

STOKES

+

u H O

LASER EXCITATION

0

500

1000

RELATIVE WAVENUMBER cm"' Figure 36 Typical spectral response of a holographic notch filter.

Lippmann (1891a,b), who was rewarded by a Nobel prize. Lippmann first performed interference color photography and described sophisticated experimental techniques to produce stable recording in argentic emulsions, dichromated gelatin or collodion. A good review of both early and current aspects of interference color photography and holography can be found in the proceedings of a recent colloquium (Fournier, 1991). Modern phase gratings are composed of photosensitive layers in which the index modulations are created by means of the techniques of laser holography. Consider the interference of two coUimated, coherent light beams which interact with a plane photosensitive film whose thickness, typically 20-60 ixm, is much greater than the wavelength of the light. A chemical developing process produces a spatially modulated index or absorption of the film medium. The resulting spectral response exhibits a narrow rejection peak which is well suited for the requirements of laser-light rejection in Raman experiments (Yang et aL, 1991; Carraba et aL, 1990; Kim etal., 1993). As recalled previously for conventional interference filters, the holographic filter can be used either in transmission or reflection, with complementary spectral responses. The holographic filter therefore constitutes an excellent dichroic beamsplitter which typically reflects 90% of the laser excitation and transmits 90% of the Raman-scattered light. The contrast ratio of laser rejection is better than 10"^. A typical curve, as shown in Fig. 36, presents

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a flat response up to 4000 cm~^ However, it suffers from a progressive attenuation of the low frequencies. A selection of the best filters is proposed by the makers to provide a sharper cutoff. The holographic filter has offered, indisputably, a major breakthrough in simplifying the design of small, analytical Raman instruments. 5. Optical Coupling of Spectral Analyzers with a Microscope The question which arises in the design of a Raman microspectrometer is how to optimize the coupling of all of the optical elements, from the sample to the detector, so as to maximize the product L31 of lesolvance by throughput [see Eq. (56)]. At the same time the stray fight background must be kept to the lowest possible level. In practice the useful resolving power needed for most analytical applications is of the order of 91 = 20 000, which corresponds to lcm~^ in the mid-visible spectral region. As recalled above, the stray light figure is improved in aU recent instruments by treating the main spectral analyzer separately from the laser-light, rejection-filter stage. However, special attention must be paid to avoid overfilling the optical components by the light beams, together with a careful positioning of the light baffles designed to eliminate spurious reflections. The invariance of the geometrical extent U along the Ught path in an instrument has a clear significance. It means that the throughput is hmited by that section of the instrument which possesses the smallest geometrical extent. In the particular case of a Raman microprobe system, the limiting extent U^jn is always imposed by the optics of the microscope. Typically, the output pupil of a microscope objective has a diameter of 3-10 mm. The image is formed at a distance which varies from 50 to 200 mm, on a slit or a spatial filtering pinhole whose dimension does not usually exceed 100 ixm. Thus, the available spatial discrimination is preserved. From the argument present above it is apparent that it is useless to couple a Raman microscope to a spectral analyzer which possesses a greater etendue. Such a combination cannot improve the throughput of the instrument. This is, then, the fundamental reason why dispersive spectral analyzers are generally preferred to interferometers in the apphcations considered here, as the intrinsic superiority of the latter instruments in terms of high resolution and geometrical extent cannot be exploited in micro-Raman spectroscopy (see, however. Section VI of this chapter).

V. PHOTOELECTRIC DETECTORS This section will begin with a brief summary of some of the more important features of single-channel detectors, which have been used primarily in

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scanning, dispersive spectrometers. These elements are now only employed in scanning systems with special requirements, or more generally in interferometric instruments working in the NIR region. Thus, in the following the major emphasis will be placed on the characteristics of the multichannel, soHd-state detectors that now tend to predominate in micro-Raman systems, either for spectral analysis or Raman imaging.

A. Single-channel Detectors

Single-channel detectors include photomultiplier tubes (PMTs) and solidstate detectors such as semiconductor photodiodes. 1. Photomultiplier Tubes For a long time the photomultiplier tube was the only detector used for low-Hght level measurements with scanning Raman spectrometers. More recently this element has been advantageously replaced by soUd-state, multichannel detectors. Its use is now limited to scanning instruments which require access to the very-low-frequency region, where the performance of multichannel instruments is limited. In a photomuItipKer the basic radiation sensor is the photocathode, which is located within a vacuum envelope. Depending on the choice of the photocathode material, good spectral response can be achieved in the range 200-900 nm. However PMTs have very poor performance in the NIR region. For each photon striking the photocathode, photoelectrons are emitted and directed to a dynode within the envelope. A number of secondary electrons are emitted at this dynode for each impinging primary photoelectron. These secondary electrons are directed toward a second dynode, and so on, until a final gain of the order of 10^ is achieved. The electrons from the last dynode are collected by an anode, which provides the signal read-out current. A fundamental advantage of a photomultiplier results from the fact that the secondary emission process contributes very little to the relative noise output of the tube. The amplification which results from the secondary emission process is responsible for the superiority of the PMT, which can approach 'ideal' performance, as limited only by the statistics of photoemission. This internal gain (10-^-10^) permits the detection of very weak signals with a good signal-to-noise ratio (S/N). The noise is determined primarily by the statistical distribution of the photoelectrons. For a PMT such as that used for spectroscopy, the noise-equivalent power (NEP) is approximately equal to 7 X 10~^^ W at room temperature and 400 nm for a bandwidth of 1 Hz.

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For low-light-level measurements, as encountered in Raman microanalysis, two basic detection techniques can be used, namely: (i) Charge-integration, in which the output photocurrent is obtained by the integration of the anode pulses which originate from the individual photoelectrons. (ii) The digital technique of photon counting, in which individual pulses are recorded. At very low light levels the photon-counting mode can improve the S/N and is thus superior to charge integration. When the signal level is low the detection Umit is determined by the dark current of the PMT and its associated noise. At higher levels the limit to the S/N is determined by the noise of the signal (photon shot-noise). In the photon-counting mode the limit is determined by the statistics which discriminate between photoelectrons and thermally emitted electrons. Since in practice the Raman signal decreases somewhat with the size of the sample, the signal resulting from the scattered Ught in Raman microanalysis is typically very weak. In order to get an acceptable S/N with sequential spectrometers and PMTs the signal must be integrated for a long time or relatively long time constants must be employed. As a consequence, the sample has to withstand a high level of laser irradiation during the long exposures and can suffer from thermal damage or photodegradation. 2. Silicon Photodiodes One of the principal advantages of p-i-n silicon cells or avalanche silicon diodes compared to a PMT is their good response in the red and far-red regions, out to 1100 nm. However silicon p-i-n diodes have no gain and the gain for an avalanche photodiode is only of the order of 100. The NEP for a p-i-n silicon photocell may be of the order of 2 x 10~^^ W at 900 nm for a 1 Hz bandwidth. The NEP of a silicon-avalanche photodiode is about 10"^'* W at 900 nm for a 1 Hz bandwidth. As a consequence, the latter detector may be an alternative to PMT detection when red excitation (a Kr"^ laser for instance) is used, since it can produce a better S/N in this spectral region due to its higher detection efficiency. 3. Germanium and InGaAs

Photodiodes

These single-channel, solid-state detectors have extended response in the NIR region from 900 nm to 1600 nm. They are thus the choice for fluorescence-free Raman spectroscopy with 1064 nm Nd:YAG-laser excitation (see Section VI). They are extensively used with FT Raman spectrometers in the NIR region, where higher throughput and multiplex advantage of Michelson interferometers is advantageous. Analyses with the

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use of dispersive instruments and single-channel NIR detectors are time consuming and cannot compete with FT Raman instruments. The Uquid-nitrogen-cooled germanium photodiode is currently the best NIR detector for both conventional scanning and FT Raman spectroscopies with Nd:YAG-laser excitation. At -196°C its NEP does not exceed 1 X 10~^^WHz^^^ and its extended spectral range enables detection of Raman shifts at about 3500 cm" ^. However its extreme sensitivity to cosmic radiation requires additional electronic circuitry to filter out intense muon pulses. At room temperature the InGaAs-photodiode detector provides the same detection level as does the Ge detector.

B. Multichannel Solid-state Detectors Low-noise, multichannel soUd-state detectors have become available in the past few years. Most of these detectors are based on silicon technology and thus are sensitive to photon energies from the near-UV region, around 280 nm, out to 1 jam in the NIR region. The bandgap of silicon defines the long-wavelength cutoff at 1.1 ixm. Other devices are based on different semiconductors such as germanium- or gallium-indium arsenide (InGaAs), whose responses extend further into the NIR region. The 1-2 jxm band is of recent interest, as its primary importance in Raman spectroscopy is its effective fluorescence rejection (see Section VI). In this chapter three different multielement solid-state detectors that can be used in the field of Raman microscopy from the UV region to 1.7 |xm in the NIR will be discussed, namely, (i) Charge-coupled devices (Si CCD), (ii) Charge-injection devices (Si CID), and (iii) Self-scanned, photodiode arrays [silicon (visible), germanium or InGaAs (NIR)]. Both CCD and CID structures employ the same basic principle: the utilization of a p-n-junction photodiode in an integrating or storage mode. 1. Integration Mode of Solid-state Detectors When photons of sufficient energy strike the siUcon substrate (Ge or InGaAs) they can create hole-electron pairs in the silicon lattice. These two charges will quickly recombine unless they are separated by the appUcation of external potential fields. In solid-state sensor a series of conductive electrodes overlying the silicon is used to create the electric fields needed to separate and store either the photogenerated holes or electrons.

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The integration mode is based on the principle that if a p-n junction is reverse-biased and then open-circuited, the charge stored on the depletionlayer capacitance decays at a rate which is proportional to the incident illumination level. By monitoring periodically the charge required to re-establish the initial voltage condition, a signal which is directly proportional to the incident illumination level can be obtained. Thus, the photon-generated current is directly proportional to the illumination level. The quantity of charge removed in a given interval of time is directly proportional to the integrated illumination taken over that interval.

2. Storage Mode of Solid-state Detectors Two functional elements are necessary to obtain charge-storage operation, namely: (i) A charge-storage element [this element can be a p-n-junction diode, as used in photodiode arrays, or a metal-oxide semiconductor (MOS)induced junction in charge-transfer devices]; and (ii) A charge-reset and -sensing circuit.

C. Two-dimensional Charge-transfer Devices Charge-transfer devices (CTD) are silicon-based integrated circuits. They consist of one-dimensional or two-dimensional arrays of MOS capacitors that behave as Hght sensor elements. Each individual cell is used to convert the incident photons into electrical charges that are stored on an electrode. The number of photon-generated charges in a given cell is proportional to the number of incident photons that strike this detector element. The CTDs have been developed with the use of two different technologies: charge-coupled devices (CCDs) and charge-injection devices (CIDs). These devices differ in the read-out technique employed. In the intercell read-out mode used in CCDs, the read-out process is accomplished by shifting the signal charge in series from one cell to another to a charge-sensitive, on-chip amplifier. In the intracell read-out mode of CIDs, the charge is transferred to a sense node in another region of the sensor element. Both of these types combine high responsivity over a broad spectral range from the soft X-ray region to the very-near IR. They are further characterized by a large number of detection elements and very low dark current. These devices are rapidly replacing other camera systems in a wide range of imaging apphcations, as well as the single-element detector in spectroscopic installations.

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1. CCD Detectors Charge-coupled devices are available in a large variety of size and format, depending on their appHcation. The CCD detectors for low-light-level scientific appHcations, which are based on high-performance chips, run more slowly than video rates and must be cooled to very low temperatures. Today, the largest CCD available incorporates over 16 000 000 pixels (4096 by 4096 elements) on an active area of 30 cm^. Although these characteristics are impressive, they still do not rival the spatial resolution of astronomical photographic emulsions.

(a) Architecture and read-out Individual regions (pixels) of a silicon substrate are delimited by a series of overlying conductive electrodes (gates) which are used to create potential wells where photon-generated charges can be stored and manipulated. The CCD comprises a parallel register (imaging region) arranged in n columns of m pixels and a series register parallel to the rows which consists of n cells. The signal charges are shifted along the columns of the parallel register by changing the bias voltage of each gate. Numerous electrode configurations have been tried, from two-phase to four-phase structures. However, the basic principle of a CCD detector can be simply described with a three-phase gate arrangement. In this configuration every pixel is subdivided into three regions held at different potentials by three overlying electrodes whose voltages can be sequentially varied. When a CCD is exposed to radiation, only the central gate of every pixel is turned on so as to form a potential well where photon-generated charges are maintained and accumulated during the exposure. Once the integration period is terminated, the photon flux striking the detector is blocked by a mechanical shutter and the bias voltages applied to the gates are modified so as to transfer the signal charge, one row at a time, across the device to the read-out node. For every pixel of a column of the imaging region, the electrode adjacent to the central electrode is turned on to the same potential; thus, a new potential well is created. The signal charge packet will be uniformly distributed underneath the two electrodes. Then, when the central electrode is turned off the signal charge is transferred completely to the adjacent electrode. During the next transfer cycle the charge packets flow from the last gate of a pixel to the first gate of its neighbor. At the same time the charges contained in the n pixels of the last row of the parallel register are shifted into the series output register. Charges in the series register are shifted in the same way, one cell at a time, to the output node located at a corner of the CCD, where they are sensed by the on-chip amplifier. The transfer

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process in the imaging area starts again only when the series register has been completely emptied. The read-out continues until the most remote charge packet of the imaging region has moved to the output ampHfier. The read-out noise is due to the uncertainty in the number of charge carriers contained in each charge packet. The read-out noise floor is about 2 electrons (e) for the best scientific grade CCD operated in the slow-scan mode (read-out rate of 40-50 kHz) with double correlated sampUng. There is a trade-off between read-out noise and readout rate because when the read-out rate is increased the read-out noise also increases. As pointed out above, scientific CCDs are read out slowly to reduce the associated noise. Thus, the complete transfer process can take several seconds for large devices. (b) Binning Instead of being read out individually, pixels can be binned within the chip to increase the S/N at the expense of spatial resolution. The principle of the on-chip binning process is to combine the charge packets from several adjacent pixels into a superpixel, prior to the read out by the output ampHfier. Parallel binning is achieved by transferring several rows in the serial register before shifting the charges in the output node. The number of rows that can be combined is limited only by the full-well capacity of the series register. Series binning is obtained by summing several charge packets in the output node before they are sensed by the output ampHfier. The number of pixels that can be joined serially depends on the capacity of the output node. Binning of pixels in two dimensions is achieved by combining the parallel and series binning processes. If it is assumed that the dark current is negligible and that the charges are added without any additional noise, two conditions are generaUy appHcable to low-light level, scientific-grade CCD detectors, namely, (i) The S/N obtained by averaging n pixels in computer memory is given by S/N = {nS)l{S-\rR^), where S is the number of photon-generated charges per pixel and R is the read-out noise of the detector. By directly binning these n pixels within the chip, the signal-to-noise ratio becomes equal to S/N = {nS)l{nS-\-R^). Thus, binning offers an advantage over averaging only in the case where S«R^ln. When this condition is fulfilled the S/N is improved by a factor of n^^^. This result can be used to improve the detection of extremely weak Raman bands that extend over a large number of pixels of the CCD. Despite the lack of S/N improvement in the case of more intense signals, the binning process provides a speed advantage that reduces the overaH read-out time. (n) Subarray read-out is an additional read-out mode that can help to optimize the S/N. It consists of restricting the number of pixels that must

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be processed by the data system to only those that contain useful information. It is employed both to increase the read-out speed and to decrease the data storage and processing time. (c) Quantum efficiency (QE) Silicon CCDs have the broadest response of any detector in the wavelength region from 0.1 to 1000 nm. Typical front-sided, illuminated imagers have a peak QE of 30-40% in the 650-700 nm region. They can respond to photons with energies greater than 1.14eV (the silicon bandgap) at room temperature, which corresponds to wavelengths less than 1100 nm. At the other end of the spectrum, they are rather insensitive to UV radiation because the overlying electrodes attenuate these photons. One method of improving the short-wavelength response of a CCD is the vacuum deposition of a very thin phosphor coating on its front surface. This coating down-converts incoming UV radiation into longer-wavelength, visible light, where the quantum efficiency is optimized. However, a large number of the down-converted photons are emitted in directions away from the surface. As they are not collected by the CCD, the QE in the UV region is less than the peak QE in the visible region. A means of improving the overall QE of a CCD is to illuminate the device through its rear surface, that which is opposite to the gate structure. However, in this configuration the substrate has to be thinned to around 10-20 luum; otherwise the electron-hole pairs are generated too far away from the CCD's potential wells and the responsivity is very poor. On the other hand, a too-thin CCD will exhibit poor visible and red QE because these photons pass through the device without being absorbed. Properly designed backilluminated devices with antireflection coatings to reduce reflection losses can achieve quantum efficiencies of the order of 70% at 650 nm and greater than 50% in the 200-400 nm range. (d) Charge-transfer efficiency For a 1024 x 1024, three-phase clocking device there are 6144 transfers (2048 elements with three gates per element) in both the parallel and series register before charge from the furthest corner pixel reaches the output ampHfier. The charge-transfer efficiency (CTE) expresses the probability of transferring correctly the charge packet from one pixel to the next. A high-quality CCD can achieve a CTE of 0.999 99 or better per transfer. This figure corresponds to a total transfer efficiency of (0.999 99)^^^4= 0.94. Thus, only 6% of the charge in that last corner pixel is left behind and lost. Recently, CTEs of 0.999 999 99 have been reported.

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(e) Dark current The dark current in a solid-state detector is a thermally generated signal due to statistical fluctuations of charges in the bulk silicon of the device. In CCD detectors, the dark current has three main origins: (i) thermal generation in the silicon substrate, (ii) thermal generation in the depletion region and (iii) imperfections in the top and bottom layers of the silicon dioxide insulator. Defects in the Si-Si02 interface can result in electronic defects called interface or surface states, whose contribution dominates the dark current in most multiphase CCD detectors. Dark current generation is especially a problem at room temperature. Cooling the device reduces the dark current by a factor of two for every 7-8°C decrease in temperature. A typical dark current of about 3.5 e pixel~^ s~Ms achievable by thermoelectric cooling at -60°C. Such a rate seems insignificant for most applications, where the pixel charge packets are read individually. However, when a large number of pixels are binned, their dark charges are combined, resulting in a large dark current. In this situation ultra-low dark currents, well below 1 e pixeP^h"^, can be obtained with the use of cryogenic devices at the temperature of Hquid nitrogen. A method called multipinned phase (MPP) has been developed in the past few years to eliminate the dark current generation at the Si-Si02 interfaces. During exposure time all of the clocks of the image zone are biased at a negative voltage, resulting in potential inversion in the substrate. This arrangement creates a large concentration of holes at the Si-Si02 interfaces so that signal charges are sent away from the interface. As a result, the interface-state, dark current contribution is cancelled and only the minor bulk contribution remains. A typical dark signal reduction of 25x is achieved in the MPP mode compared with conventional operation. The MPP method reduces the need for cryogenic cooling, since low dark currents are achievable with the convenience of a thermoelectric-cooled system. An advantage associated with this technique is the reduction in the full-well capacity of the device. The MPP technology has proved to be beneficial in other areas of CCD performance related to the front surface. (f)

Blooming

The spilling of charge from an illuminated detector element to adjacent elements is referred to as blooming. Many commercial CCDs are equipped with antiblooming drains to minimize the effect of charge blooming. (g) Drawbacks of CCDs Like other siUcon devices, CCDs respond to cosmic rays. Each high-energy particle creates thousands of hole-electron pairs that interfere with the

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spectra or images being recorded. The CCDs are inherently slow detection systems which are not well adapted to signal optimization at the beginning of a recording. Thus, they are not suited for time-resolved spectroscopy. For applications to kinetic studies, a CCD detector can be coupled to an image-intensifier tube in order to benefit from its gating capability. (h) Advantages of CCDs In multiple-input spectroscopy, the simultaneous acquisition of spectra can be accompHshed by focusing individual sources either optically or fiberoptically along the entrance slit of the spectrometer. Practical limits to the number of sources are from two to ten. 2. CID Detectors Charge-injection devices differ from CCDs in several respects, namely, (i) Semiconductor junctions used to collect photogenerated charges consist of n-doped silicon grown on a p-doped substrate; thus the photogenerated charges are holes instead of electrons. (ii) A photogenerated hole packet always remains within the detector cell where it was collected, rather than being sequentially moved from one cell to the next until it reaches the on-chip sensing node. (iii) Charge measurement can be achieved in two ways: destructive read-out by injection of the photogenerated charges into the bulk silicon substrate, where they recombine, or nondestructive read-out by moving the charge back and forth between two MOS charge-storage capacitors included within each individual pixel site. Either process can be repeated several times to reduce the uncertainty in measurement. In general, pixel read-out noise is reduced by the square root of the total number of times the read-out is repeated, as this noise is Gaussian. Data can be extracted randomly and nondestructively from individual pixels, multiple sub arrays, or from the entire frame (detector, imager). Charge information is measured in the detector element where it is collected, rather than moved to an on-chip ampHfier. The photogenerated charge never leaves the detector element where it is collected, so the binning process is not available with CIDs. A CID allows the charge information to be read nondestructively (NDRO). The quantity of charge can be measured without removing it from the detector element. The charge is shifted back and forth underneath two crossed electrodes. Nondestructive read-out can also be employed to extend the useful dynamic range of the sensor. This function is obtained with the use of random-access integration in which different exposure times can be used for

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different regions of the CID. This procedure allows the acquisition of both intense and weak signals in the same exposure cycle. Once a desired signal level has been reached in a given region, the accumulated charge is injected into the substrate. The integration is then reset so that the region is read out repeatedly throughout the measurement cycle. Other detector elements in which weak charges are collected can be left undisturbed, permitting integration to be made when a suitable S/N is reached. The abihty to vary independently pixel integration times, depending on the incident flux, extends the useful dynamic range of this detector. The CIDs have high video-line capacitance, which results in a much higher read-out noise than that of CCDs, even when multiple, nondestructive read-out is employed. With the use of random-access integration, different exposure times can be used for different regions of a CID with the exposure times determined during the exposure of the CID to the source. The inherent noise in a scientifically operated, cooled slow-scan CCD read-out, which is typically less than 15 electrons, is lower than that in a CID. However, the nature of the CID allows a single, randomly addressed pixel to be read repeatedly. Thus the noise levels can be reduced with the use of signal averaging, both during and after exposure. The CIDs have certain advantages, for example when Raman features vary greatly in intensity it is necessary to evaluate extremely high, as well as very low, light levels during a single analysis. Thus, the detector must be used with highly variable periods of integration. On the other hand, CCDs are not readily amenable to this type of analysis because the full image frame must be flushed upon read-out. Hence, weak-signal integration is terminated before an adequate S/N is reached if strong lines are to be quantified before their associated detector elements saturate. At the other extreme the quantification of weak signals leads to saturation of strong ones. Thus, CIDs have not found as widespread a use as CCDs in the field of Raman spectroscopy. However, they are sometimes useful because they can be read with random access.

D. Self-scanned, Photodiode-array Detectors 1. Visible Photodiode Arrays

(PDA)

Unlike PMTs, photodiode arrays can acquire data in a multiplex fashion. That is, information at many different wavelengths can be recorded simultaneously, thus reducing the time required to complete a spectroscopic experiment and/or improving the S/N. The S/N of a typical diode-array pixel is much lower than that of a PMT (often by up to three orders of magnitude). This characteristic is due to the

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unit gain and high dark current noise of the photodiode. Consequently, these arrays cannot be used normally as a direct detection system in low-hght-level applications such as Raman spectroscopy. However, they can be employed in low-light-level measurements by incorporating an image intensifier to boost the overall gain. A monohthic, self-scanning photodiode array consists of a row of siUcon photodiodes, each with an associated storage capacitor on which the photocurrent can be integrated, as well as a multiplex switch for periodic read-out via an integrated, shift-register scanning circuit. In this mode, although there is no front-end gain, advantage can be taken of the very high quantum efficiency response of siHcon diodes. The responsive quantum efficiency reaches a maximum of about 80% at wavelengths from 700 to 900 nm and is greater than that of any conventional photocathode over the entire 0.4-1.1 |xm region. (a) Operation These elements are composed of from 128 to 2048 siUcon-diode sensor elements of 2.5 mm in length and arranged on 25.4 ijim centers with no dead space between adjacent diodes. By reverse-biasing the diode to 5 V (bias of 5 V between the substrate and both dummy and active video fines), a charge can be stored on the capacitance associated with each diode, which then floats. Electron-hole pairs generated in the diodes due to incident photons (the signal) and to thermal effects (dark current) will slowly discharge the diode capacitance until some specified integration time has elapsed. At this point each diode in turn is again reverse-biased to 5V. The charge required to rebias is then a measure of signal plus dark current. The resultant recharge pulses are carefully monitored with charge-sensitive preampfifiers and then converted to voltage pulses, which are amplified before being converted to signals compatible with a computer. (b) Noise For a Reticon array used in the direct mode, the signals produced are weak. At low temperatures, for exposures of less than 10^ photons, the S/N in the output is limited primarily by the effect of the read-out noise. The read-out noise is associated with the capacitance of the diode array. The limiting noise sources are the reset noise and the preampfifier noise. The reset noise is given by {lle){kTC^^'^, where C^ is the diode capacitance and e is the electronic charge. It is independent of bandwidth and, furthermore, sets the lower limit to the noise in a given detector. It represents the inherent uncertainty of restoring the charge in a given diode.

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The preamplifier noise is directly proportional to the capacitance of the array (video line capacitance). The high output capacitance is the chief drawback of the MOS photodiode array, as compared with the CCD. For a self-scanned, photodiode array the capacitance Cy of a given video line is high (~100pF), since it is the parallel sum of the capacitances of all the field-effect transistor switches connected to that video line.

2. Intensified Photodiode Array

(IPDA)

For applications, such as Raman microspectroscopy, which require operation at very low light levels, the best results are obtained by intensification of the incident radiation before it strikes the diode-array detector. This image intensification requires photoelectric tubes with a photoemissive photocathode and a high-gain, photoelectron ampHfier. In most installations a proximity focused, microchannel plate (MCP) intensifier tube is coupled to an external photodiode array by optical fibers. The intensifer tube contains a photocathode in a sealed glass envelope, a stack of two or three microchannel plates and a phosphor screen deposited on a fiberoptics plate. Photons striking the photocathode are converted into photoelectrons, which are amplified by the microchannel plate by a factor of about 3000. The phosphor screen converts the ampHfied photoelectrons back to photons, which are then detected by the photodiode array.

3. Intensified Photo diode-array Detectors versus CCDs The performance of an IPDA detector is limited by the microchannel plate image intensifier used. The QE is reduced to 10%, with a much more restricted response in the far-red region. The MCP also degrades significantly the spatial resolution of the system. Precision photometric performance is difficult to achieve because of nonhnear response and image persistence. The MCPs are also susceptible to damage by light overload in a way that the CCDs are not. Intensified photodiode-array detectors remain as alternatives to CCD detectors for signals that do not require very long integration times. They are also very useful for signal optimization, since they can reflect in real time any signal variation - for example that which is produced when the sample is moved under the microscope objective in order to find the best location, or that due to laser adjustments. The IPDA detectors also have a great advantage in time-resolved spectroscopy. The MCP intensifier tube can be gated very quickly, so that only photons arriving when the tube is switched on are monitored by the diode array.

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E. NIR Photodiode Arrays and Solid-state Detectors Other internal, photoemission detectors that are responsive to wavelengths longer than 1000 nm are now being manufactured, e.g. platinum silicide (PtSi) Schottky-barrier detectors, InGaAs, InSb, germanium, MCT (HgCdTe). Some of these devices are outside the scope of this chapter. Thus, only those which are appHcable in Raman spectroscopy - germanium and InGaAs array detectors - will be discussed here. Unlike visible photodiode arrays, NIR multiplex detectors are used without intensifier tubes and low-Hght-level detection can be achieved only with long integration times. 1. Germanium Photodiode

Arrays

These hquid nitrogen-cooled, multichannel detectors show good sensitivity from 900 to 1600 nm and are well suited for NIR excitation between 980 and 1064 nm. This type of detector is called a hybrid device because the sensor and the read-out circuit are made of different materials - germanium and siHcon, respectively. Arrays with 128 or 256 pixels are now available. The size of each element varies from 0.09 x 2.5 mm^, arranged on a 100 \im center-to-center distance, to 0.05 X 0.3 mm^ with a 50 juim pitch. A long exposure time is preferable to multiple accumulation because the read-out noise is high; the integration time varies from a few seconds to 400 s. These detectors offer the multiplex advantage. The multiplex Ge detector can be considered to be the first commercially available NIR detector that permits fluorescence-free Raman recording (see the following section). However, it has a cutoff wavelength at about 1.45 |xm, which precludes the detection of the C-H region at 3000 cm~^ when excitation at 1064 nm is employed. The best system compromise would consist of a germanium detector combined with a laser at 980 nm (a diode laser or a tunable Tiisapphire laser, for instance) that would allow the observation of the entire Raman spectrum without sacrificing the C-H stretching region of the spectrum. 2. InGaAs Photodiode Linear Arrays and Two-dimensional

Matrices

The InGaAs detector is sensitive from 0.9|ULm to 1.7 |xm and is latticematched to an indium phosphide substrate at room temperature. The lattice matching minimizes the defect density at the interface and leads to low dark current at room temperature. The response is greater than 30% at 0.9 |xm and more than 65% between 1 and 1.6 (xm. At room temperature it exhibits a Z)* of 1 X 10^^ and at 230 K a D* of 3.5 x 10^^ cm H ^ W ' ^ Linear arrays with 128 to 512 individual photodiodes are commercially available and consist of rectangular sensors (30 ixm width x 100 jjim aperture height with a 50 jxm

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center-to-center distance). Generally, a two-stage Peltier cooler maintains a stable operating temperature as low as -40°C with -10°C liquid-cooling assistance. Two-dimensional matrices are now available. They consist of a 128 x 128 element, InGaAs photodiode array which is integrated with bump-indium technology to a silicon complementary metal oxide semiconductor (CMOS) read-out multiplexer. The photodiode array is back-illuminated to provide a 100% optical fill factor.

F. Other Two-dimensional-imaging Detectors: PIVITs These detectors are not classified as solid-state detectors, since they incorporate a photoemissive sensor, as do PMTs. Each photoelectron released from the photocathode is amplified by a set of microchannel plates to produce a cloud of about 10^ electrons. The electron pulse is accelerated to strike a semiconductor, position-sensitive detector. The electrons are detected at the four corners of a resistive anode and the centroid is determined to within 70 |xm in both the X and Y directions by a position analyzer. The pulse amplitude, which is determined by summing the current from the four corners, is used to discriminate against low- and high-current events, such as those arising from the microchannel chip current or cosmic rays. With respect to discrimination, the imaging PMT and the traditional, photon-counting devices are similar. The dark count summed over the full one-inch (2.54 cm) diameter of the detector is about 10 counts s~^ (cps), when the detector is held at -30°C with a total bias voltage of 4200 V. Given a linear spatial resolution of 400 fine pairs for this two-dimensional detector, these conditions yield a dark count of 0.025 cps per channel, or about 90 counts h~^ per channel. With this dark count, optical signals with a peak intensity of 0.005 cps can be observed with this detector. Photon-counting imaging systems can be classified as integration types or real-time (nonintegration) types. In integration systems the charge signal is accumulated on the target of the detector, which is subsequently read out. This type of system is generally associated with phosphor-output, image intensifiers. Nonintegration types process the signal in real time and generally employ an anode, which provides an electrical output. The anodes are continuous resistive films with electrodes positioned at their periphery to carry out the charge-division calculation. Carbon-based, as well as silicon-based, position-sensitive device (PSD) anodes can be used. Silicon-based devices offer an additional gain factor to that of an MCP cascade alone. The charge signal corresponding to a single incident photon is distributed to the four electrodes, amplified and fed to the position analyzer. The

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positions of the photons are calculated by a charge-division method that determines the centroid of the electron charge. The maximum count rate of the position analyzer is about 4 x lO^cps for random time of photon arrival. Thus, this type of detector is not well adapted to the Raman analysis of even low-fluorescent samples, since in that case the high incident photon rate due to the fluorescent background can very rapidly saturate the position sensor. As a consequence, imaging PMTs are no longer employed as two-dimensional detectors in the field of Raman spectroscopy. They have been completely superseded by high-performance and low-cost CCD detectors.

VI. FLUORESCENT SAMPLES A. Introduction The analytical application of Raman spectroscopy can be severely restricted by laser-induced fluorescence. This problem is still present in Raman microanalysis. However, the micromethod offers the possibihty of choosing within the sample - via the microscope - the region of interest which exhibits the lowest fluorescence emission. This technique is especially useful in the case of inhomogeneous samples, where the fluorescence is not evenly distributed within the sample. However, when the fluorescence emission emanates from the region of interest, an alternative approach must be employed to reduce the fluorescence background. A number of techniques have been proposed for minimizing fluorescence interference in Raman spectroscopy. Among them are: (i) Exposure of the sample to the laser irradiation for a long period of time in order to burn out the fluorescence emission (photobleaching). (ii) Temporal discrimination between fluorescence emission and Raman scattering. In the latter method pulsed sources and gated detectors can be advantageously employed to separate Raman scattering, which is practically instantaneous, from the long-lived fluorescence or luminescence. When the detection gate is opened during the hght pulse, the photodetector integrates the Raman signal; but when it is closed, all of the signal originating from the fluorescence emission of the sample is eliminated. In practice, the hmitation to single-pulse Raman experiments is imposed by the threshold of nonlinear processes and dielectric breakdown. Excitation by a laser pulse of moderate energy (100 |xJ) in the ns range is barely tolerable for a defocused beam illuminating 1 mm^ of sample (corresponding to a local irradiance of l M W c m ~ ^ for a 10 ns pulse). Such a short pulse would

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certainly destroy a microsample, since the beam focused down to an area of a few jjim^ will produce a local irradiance of several TWcrn"^. In principle, the time-resolved technique offers an elegant means to discriminate physically Raman scattering from fluorescence or luminescence. Unfortunately, in practice it is incompatible with Raman microprobing at high spatial resolution unless the peak power is attenuated and a large number of pulses is accumulated by the detection system, in order to improve the signal-to-noise ratio. (iii) The use of nonvisible laser excitation. Unlike fluorescence emission, Raman spectra possess the inherent advantage that they are not localized in a given spectral region. Depending on the wavelength of the exciting radiation, they may be produced over a broad range which extends from the UV to the NIR. Near-UV laser excitation can be used to take advantage of resonance enhancement of the Raman signal. In this case the fluorescence is not eliminated, but the enhanced Raman signal may become stronger than that of the fluorescence emission, and thus can be detected more easily. However, attention must be paid to the level of laser irradiation, as it must be kept low enough to avoid local degradation resulting from the absorption of Ught by the sample. To discriminate against the fluorescence background in spontaneous Raman experiments there is another approach, i.e. to excite the sample at a wavelength that is longer than that which corresponds to the lowest energy, electronic absorption band. The first use of extreme-red excitation for Raman analysis was made by Stammreich and Forneris (1961), before the advent of laser sources and sophisticated instrumentation. With the use of discharge lamps (helium and rubidium vapor) as monochromatic sources and detection with photographic emulsions, they demonstrated the feasibility of recording the Raman spectrum of deeply colored samples such as liquid bromine. The use of long-wavelength excitation is fundamentally unfavorable due to the decreased scattering factor and the reduced sensitivity of photodetectors with increasing wavelength. Hence, the prime motivation for the use of NIR excitation in laser Raman instruments is the production of fluorescencefree spectra. In the past few years various techniques for fluorescence rejection based on NIR excitation have been developed. In this section the potential of the NIR excitation method in the field of Raman microanalysis will be discussed.

B. NIR Raman Microanalysis The principal advantage of the use of NIR exciting radiation is that the energy of light quanta in this spectral range is too low to excite fluorescence spectra.

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However an inherent drawback of this technique is the v^ dependence of the intensity of Raman scattering [see Chapter 1, Eq. (7)]. With laser excitation at 1064 nm, the resuh is a decrease in the Raman signal by a factor of 18 compared with the case of excitation at 514.5 nm. Another limitation is due to the lower sensitivity of NIR detectors compared to their visible counterparts (see the previous section of this chapter). The first experiments on fluorescence rejection with the use of NIR radiation were carried out by Hirschfeld and Chase (1986). They employed Nd:YAG laser excitation at 1064 nm and a Fourier transform spectrometer to detect the Raman spectrum from a macrosample. The choice of this system was based on two important features of interferometers, namely high throughput and simultaneous detection of a large number of spectral elements (see Section IV of this chapter). It was employed to compensate for the inherent weakness of the Raman signal in this region. However, another method is now available which virtually ehminates fluorescence interference which would occur with most samples under visible excitation. It is a conventional dispersive technique - either scanning or multichannel - which employs NIR-sensitive detectors, as well as laser excitation beyond 1 fxm. C. FT Raman Spectroscopy

An FT Raman instrument consists of a continuous-wave Nd:YAG laser as the source and an NIR-optimized Michelson interferometer fitted with either a Ge or an InGaAs detector. Unlike grating spectrometers, the entrance aperture of an interferometer is not a narrow slit, but a larger, circular aperture called the Jacquinot stop. This aperture is necessary to Hmit the beam to those rays which can interfere, depending on the spectral resolution (Schrader et al., 1994). In an interferometer, the theoretical resolving power is equal to twice the maximal displacement of the moving mirror, or maximum path difference (MPD), divided by the wavelength of the radiation to be analyzed. This quantity corresponds to a spectral resolution of ArP = 1/(2MPD). In order to make use of this resolving power the maximum radius of the Jacquinot stop is determined by r ==/(A/MPD)^^^, where / is the focal length of the collimator mirror. For a typical resolution of 4cm~^ at 1064 nm, the resolving power is approximately equal to 2500; and, for a 300 mm colhmator, for instance, the entrance aperture is 8 mm in diameter. Typically, this larger area corresponds to a throughput gain of 20 compared with a grating monochromator operating with the same spectral resolution. The throughput advantage (or Jacquinot advantage) of the interferometer can be obtained only if the beam of light entering the interferometer (Raman signal) completely fiUs both the Jacquinot stop and the beamsplitter. Then, the image of the Jacquinot stop can match exactly the size of the detector.

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This condition results when relatively large samples are employed, which explains the great success of FT Raman spectroscopy with respect to dispersive techniques for the study of bulk materials. In this case the large scattering volume can partially compensate for the loss of Raman intensity due to the v^ law. However, the interferometer becomes less advantageous in the appHcation to Raman microanalysis. Although some authors have tried to demonstrate the capabilities of FT Raman spectroscopy for the investigation of small samples without the use of a microscope attachment (Williams, 1990), it is now generally conceded that the analysis of jjim-sized samples requires the use of high-magnification, large-aperture objectives Uke those employed in conventional microscopy. However, such microscopes have a restricted throughput compared with that of an interferometer. For microscopic samples the image of the Jacquinot stop is typically a few hundred jxm in diameter in the sample plane. Thus, for very small samples whose dimensions are less than this value, the throughput of the interferometer is not fully used with a microscope attachment and the Jacquinot advantage is not completely exploited. Another advantage of an interferometer is the simultaneous detection of all wavelengths of light entering the device. This characteristic is known as the multiplex or Fellgett advantage, which leads to a vastly superior S/N than that achieved by scanning instruments (this advantage is analogous to the multichannel gain observed with dispersive instruments fitted with multielement photodetector arrays). However, unlike the multichannel advantage, the Fellgett advantage applies only if the performance of the instrument is limited by detector noise. Otherwise it may be completely lost, or at least become disadvantageous, if the instrument is shot-noise limited (Hendra et al., 1991). This situation can restrict the performance of an FT Raman microscope, because in practice the intensity of the Raman scattering decreases somewhat with the size of the sample. Thus, low-noise detectors are required for the detection of very weak signals. In spite of these limitations, several groups of investigators have successfully demonstrated the feasibility of an FT Raman microprobe for recording fluorescence-free Raman spectra. A conventional microscope equipped with glass objectives was employed (Bergin and Shurvell, 1989; Messerschmidt and Chase, 1989; Sawatzki, 1991; Sommer and Katon, 1991, 1993; Sawatzki et al., 1994). Such an FT Raman microscope accessory has now been commercialized by Bruker. It consists of a conventional optical microscope coupled to the laser and the interferometer via NIR-glass fiberoptics (Turner, 1994). Spatial resolution in instruments of this type is limited by the intensity of the signal collected by the detector - more precisely by the S/N at the detector. Although an ultimate spatial resolution of 4 ixm has been claimed in the hterature generally at the expense of S/N and/or measurement time (Sommer and Katon, 1993), typical resolution usually ranges from 15 to

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100 |xm. Thus, it is far from the diffraction-Hmited possibiUty of conventional Raman microscopes. It has been suggested that the size of the detector could be reduced to decrease the associated noise. However, a reduction to 250 (xm may be impractical due to the difficulty in obtaining and maintaining the optical alignment of the detector (Sommer and Katon, 1993). D. Conventional Dispersive Raman Spectroscopy

Since the most important factor in the elimination of fluorescence background is the use of long-wavelength excitation, there is no reason not to employ conventional dispersive techniques rather than the FT method to obtain fluorescence-free Raman spectra. Moreover, the throughput of dispersive instruments becomes increasingly more favorable towards longer wavelengths and can partially counter the weakness of the Raman signal due to the v^ dependency of the Raman intensity. For a long time NIR grating Raman spectrometers were limited to single-channel operation due to the lack of multielement NIR detectors with extended response (beyond 1 \xm). Due to the low speed of the scanning technique such instruments were devoted to macroanalysis of fluorescent samples which could withstand a long period of laser illumination. Today, with the advent of various kinds of sohd-state array detectors, NIR Raman spectroscopy can take advantage of the multichannel technique and benefit from almost all of the possibilities of visible, multichannel Raman spectroscopy. 1. Single-channel Technique In the past few years, several workers have demonstrated the possibility of using the scanning dispersive technique to obtain good-quality fluorescencefree Raman spectra of large samples (Portfield and Campion, 1988; Barbillat and Chapput, 1990; Engert et al., 1991). Scanning instruments can even provide superior performance to FT spectrometers at low frequencies, for the detection of small Raman shifts, a region where current FTRaman instruments exhibit poor performance. However, NIR scanning monochromators, even when equipped with single-channel germanium or InGaAs cryogenic photodetectors, are inherently slow. They are thus not well adapted to the study of fxm-sized samples due to the potential damage arising from long exposure to the laser beam. 2. Multichannel Technique Multichannel detection can aid considerably in obtaining fluorescence-free Raman spectra of luim-sized samples by reducing the recording time with

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simultaneous acquisition of a large number of spectral elements. NIR multichannel, Raman microprobing of small samples has became practicable with the development of multielement detectors having a spectral response extending into the far red (siUcon CCD detector) and the NIR beyond 1 ixm (liquid nitrogen cooled Ge and InGaAs Hnear photodiode-array detectors). These detectors, which typically consist of several hundreds of individual NIR light sensors, offer a multichannel advantage, as do FT spectrometers with the multiplex advantage. Thus, the S/N of a multichannel recording is n^'^ times larger than that obtained with a scanning instrument, where n is number of simultaneously recorded spectral elements. Multichannel spectrometers typically consist of a triple spectrograph (a zero-dispersion, double-grating premonochromator coupled to a spectrograph) which provides a high degree of laser-frequency rejection and a high transmission close to the laser line (see Section IV of this chapter). This arrangement allows easy access to the low-frequency region. Recently, new designs which include holographic notch filters have been introduced to reject efficiently the laser line. This element replaces the zero-dispersion premonochromator. This modification limits the low-frequency capability of the instrument, but increases significantly the overall transmission of the instrument, which helps to compensate for the weakness of the signal. The success of visible Raman microprobing has clearly demonstrated that the small aperture of a microscope can be easily matched to the larger aperture of a grating spectrometer. A simple transfer-optical system between the microscope and the monochromator is used to adapt the corresponding apertures. Thus, the image of the laser spot on the sample is optically conjugated to the narrow entrance slit of the monochromator. The back aperture of the microscope objective exactly fills the dimension of the grating, which represents the entrance pupil of the monochromator. All of the scattering signal collected by a high-magnification, wide-aperture microscope objective can be transmitted without loss to the monochromator and hence to the detector, whose area also matches the image of the laser spot. As a consequence, NIR, multichannel Raman spectroscopy not only helps to reduce the fluorescence background, but also offers the same level of analytical capabilities as its visible counterpart. These possibilities include microanalysis, as well as access to the low-frequency region, when a high-performance grating spectrometer is employed to reject the intense exciting line. The NIR, multichannel method can also benefit from the recent development of the confocal technique, which results in an improved axial resolution and depth discrimination. Successful instrument design involves the use of silicon-based CCD detectors, whose spectral response extends up to 1 |xm (Barbillat et al., 1991). However, in order to record an entire spectrum, the excitation is limited to wavelengths below 770 nm. In this case fluorescence eHmination is not as efficient as with wavelengths beyond 1 |xm.

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So far, the most successful instrument designed for fluorescence rejection involves the use of hnear Ge-photodiode arrays at hquid nitrogen temperatures, whose sensitivity then extends in the NIR from 900 to 1600 nm. The Nd:YAG laser at 1064 nm, the diode laser at around 980 nm or the Ti/ sapphire tunable laser are well adapted to the spectral response of this detector, since the Raman spectra do not extend beyond 1.6 jxm with the longer excitation wavelength at 1064 nm. A micro-Raman spectrometer has been especially designed to demonstrate the potential of this detector (Barbillat et al., 1993) and a commercial version is now manufactured by DILOR. It consists of a conventional optical microscope coupled to a single-stage grating spectrograph equipped with a hquid-nitrogen-cooled 256-element, germanium photodiode array. The rejection of the laser radiation is performed by a holographic notch filter between the microscope and the spectrograph. A confocal arrangement provides improved spatial discrimination along the optical axis. Under these conditions, spectra of a variety of both solid and liquid samples have been obtained. With less than 50 mW of laser power focused on the sample by a 40x microscope objective, acquisition times range from a few seconds to 400 s, depending on the sample. The InGaAs-array detector might become an alternative to the Ge detector due to its thermoelectric cooling. It does not require cryogenic equipment and, furthermore, it has extended response in the NIR. The imaging capabilities of visible Raman microprobes will be extended to the NIR region as soon as two-dimensional NIR detectors become available. Such two-dimensional InGaAs-array detectors are already manufactured, but their performance needs to be optimized to suit the requirements of Raman spectroscopy.

E. Conclusion

As expected, the use of NIR excitation in Raman microscopy beyond 1 juim results in a considerable fluorescence rejection. Nevertheless, it should be noted that in some cases even NIR radiation does not overcome the fluorescence problem and occasionally a background is observed on NIR Raman spectra. Two complementary methods are now available for reducing fluorescence interference, each with its respective advantage. Fourier transform Raman spectroscopy is the better choice for the study of large samples, since this technique benefits fully from the high-throughput advantage of the interferometer. In the case of jxm-sized samples, some of the advantages of the interferometer are no longer obtained and the spatial resolution is then hmited to a few tens of jxm. For Raman microanalysis with resolution close to the diffraction limit, it is preferable to employ the dispersive, multichannel

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technique with Ge- or InGaAs-array detectors. The latter system provides the spectroscopist with nearly the same performance as that of visible Raman microprobes, as well as fluorescence rejection.

VII. FIBEROPTICS A. Introduction

A variety of optical systems have been described which use optical fibers to perform remote Raman spectroscopic analyses (McCreery et al., 1983; Schwab and McCreery, 1984; Hendra et al, 1988; WilUams, 1990; NguyenQuy Dao et al., 1992; Jiaying Ma et al., 1994). In this section, the basic features of different optical configurations will be briefly recalled and special attention will be paid to the spatial resolution capabilities of this technique. In this respect the characteristics of various optical fiber arrangements will be compared with those of conventional Raman microprobes. It is convenient to distinguish two main categories of optical fiber probe, often referred to as 'optrodes': (i) Direct-coupled, in which an optical fiber end is in contact with, or placed at a short distance from, the sample. For the analysis of Uquids, the fiber may be immersed in the sample medium. (ii) Indirect-coupled, in which the fiber is coupled to the sample via an optical transfer system consisting of lenses or mirrors. In the latter case the properties are to some extent comparable with those of a confocal microscope, in which the fiber ends play the role of confocal pinhole diaphragms.

B. Characteristics of Optical Fiber Systems

Optical fibers consist of a cylindrical core of transparent material of high refractive index rii with an outer layer or 'cladding' of lower index Ai2- Light entering the end of such a light guide can be trapped over long distances due to the total internal reflection at the core-cladding interface. Total reflection occurs for rays incident on the interface with an angle greater than the critical angle 6c defined by sin 6^ = — .

(74)

Hi

If the refraction at the end of the fiber is taken into account, it is found that only the light entering within an 'acceptance cone' of half-angle a can be

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129

^2 2 CLADDING CORE DIAMETER

AIR«o=l OR SAMPLE MEDIUM U'Q Figure 37

Total internal reflection in an optical fiber.

totally reflected (see Fig. 37). Then, sm a =

(75) no

where HQ is the refractive index of air, or of the immersion medium. Instead of using an index step between core and cladding, the same effect can be obtained in 'graded-index' fibers by employing a continuous variation of the refractive index along the radius r. Most often a parabohc relationship is used; thus. n = n^ 1

a

(76)

where rii is the index at the center, a is the external radius and fe is a coefficient which varies from 0.01 to 0.02. Of particular interest are the imaging properties of SELFOC light guides.* They are made of a graded-index rod which exhibits excellent imageformation capabilities, so that an image formed on the front end is transferred to the back end. Such a graded-index lens (GRIN), which can be used as a microscope objective, has a typical resolution of 200 line-pairs mm~^. Both stepped and graded index optical fibers are usually described in terms of the numerical aperture (N.A.) defined by N.A. = s i n a ,

(77)

where a is the acceptance angle in air (HQ = 1). It may be of interest to consider the equivalent //number or photographic aperture in order to compare optical fiber sensors with conventional macroor micro-Raman, light-collection lenses (Table 1). As recalled above (see Section III), the//number « 1/(2 N.A.). *SELFOC microlens and graded-index optical fibers manufactured by Nippon Sheet Glass Co., NSG America, Inc., 28 W^orld's Fair Drive, Somerset, NJ 08873, USA.

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Table 1 Examples of available optical fibers. Core material

Cladding material N.A. //number Half-angle acceptance, a (deg)

Doped silica glass Silica glass High-index glass

Silica glass

0.22 2.3

12.7

Polymer Low-index glass

0.27 0.55

15.6 33.3

1.9 0.9

In optical fibers with large core diameters, light can propagate in many different modes; such fibers are referred to as multimode fibers. When laser light is transmitted via such a multimode guide, most of the coherence is degraded. Thus, the light in the output section of the fiber exhibits a random, speckle distribution of intensity. Multiple bending and twisting along the fiber also tends to destroy the initial polarization of the input laser beam. However, special polarization maintaining fibers are available with a nonaxially symmetric core structure, for instance an elUptical section. Or, the asymmetry can be produced by the application of a permanent stress, permitting the propagation of a polarized laser beam without loss of polarization orientation. As the core diameter of the fiber is reduced, the number of propagation modes decreases. For core diameters of 3-9 |xm only one mode exists, so that the fiber is called single-mode or monomode. The coupling of a laser beam into a monomode fiber requires both exact centering and alignment, as well as careful mode matching. The mode structure of a single-mode fiber is quite similar to a Gaussian mode, but the core diameter is only of the order of 10 wavelengths of the light and is thus considerably smaller than that of a TEMQO laser beam.

The beam properties are usually described in terms of the asymptotic divergence, which is defined as shown in Fig. 38. The aperture angle 20Q of the emergent cone of light is given by 260 = -

^

-

^

,

(78)

where 2wo is the mode diameter. For example, a monomode fiber with a mode diameter of 2WQ = 9A exhibits an asymptotic divergence of 2^0 ^ 0.135 rad. By comparison, a typical HeNe laser in free propagation possesses a beam-waist diameter of the order of lOOOA, with a corresponding asymptotic divergence of 1 mrad. It is interesting to note that for a monomode fiber, the asymptotic divergence 20Q differs significantly from the numerical aperture, N.A. For instance, for a silica fiber N.A. = V(2nAn) = 0.12. Clearly, 2 N.A. = 0.24 is much larger than the divergence 2^o = 0.135 rad.

Instrumentation

-HS

^

131

1 2 Wo

Figure 38 Asymptotic divergence of a single-mode optical fiber.

When a laser beam is coupled into a single-mode fiber, the focusing lens has to be chosen so as to fulfill the mode-matching conditions. That is, the focused, laser-spot diameter must be equal to the mode diameter, and the angular aperture of the incident cone of Ught must be equal to the asymptotic divergence 26Q of the fiber (Jeunhomme and Monnerie, 1980; Ladany, 1993).

C. Direct-coupled Raman Sensors In order to estabhsh a comparison of the various types of Raman optrode, it is convenient first to consider separately the two functions of an optical fiber system, namely 1. Couphng of the laser-excitation fiber to the sample, and 2. Raman light collection. The final step will involve an evaluation of the figure of merit of the entire system by combining these two functions. Then, the advantages and drawbacks of each configuration will be examined in terms of: (i) Raman signal-to-background ratio, (ii) Useful sample volume, and (iii) Spatial discrimination. 1. Coupling of the Laser-excitation Fiber to the Sample When an optical fiber is immersed in a sample medium, the laser exciting beam with power P produces a local irradiance / which varies rapidly in

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LASER POWER P EXCITATION FIBER

Figure 39 The excitation cone in the sample medium.

Figure 40 Approximation for a single-mode fiber. space. For a thin layer of sample in direct contact with the fiber output of area 5, the irradiance / is maximum and is equal to PIS =

4F

(79)

if it is assumed in the first approximation that the distribution of intensity is quasiuniform for a multimode fiber of diameter D. Consider now an elementary volume of sample Av situated at a distance Z»D along the optical axis. The irradiance / decreases rapidly according to a IIZ^ law (see Fig. 39). To evaluate the distribution close to the fiber with a better approximation, it has been proposed to assimilate the light beam into a cone of radiation of half-angle a tangent to the end of the fiber (see Fig. 40). This approximation is valid for a single-mode fiber, which is equivalent to an imaginary point source located inside the fiber at a distance Zo = ro/tana (Zhong-Yuan Zhu and Yappert, 1992a). The axial distribution of the irradiance is of the form constant

(80)

and, for a Gaussian light beam, the angular distribution is given by /(z,,) = / ( z , o ) e x p ( - ^ | ^ ) .

(81)

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EXCITATION FIBER

yx Figure 41

COLLECTION FIBER

Excitation and collection efficiency of a double-fiber sensor.

In the case of a multimode excitation fiber, a multi-Gaussian distribution should be used. A review of the different approximations proposed to model the excitation field has been given by Plaza et al. (1986). The parameter K' specifies the effective radius of the Gaussian beam. 2. Raman Light Collection Similar approximations show that the light cone of solid angle ft collected by the entrance of the Raman collection fiber is maximum for a thin sample layer which is in direct contact with the end of the fiber. Again, the efficiency of collection decreases rapidly according to a 1/Z'^ law for an elementary volume of sample dv at a distance Z' (see Fig. 41). Consider the Raman signal excited in and emitted from an elementary volume du, as shown in Fig. 41. It is then defined by dv = d5 dZ = 27r(Zo + Zf ^^"^^^ '

(82)

an expression which must be integrated over the total volume of the overlapping cones of excitation and collection. As would be expected, the collection efficiency, as well as the useful volume, depend on the geometrical arrangement of the excitation and collection fibers. Three typical configurations have been extensively studied and modelled, namely (a) Single-fiber sensors, (b) Double parallel-fiber sensors, and (c) Nonparallel-fiber sensors.

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The results obtained with double-fiber sensors can be extended to multiplefiber systems in which a central excitation fiber is surrounded by a collection-fiber bundle. A complete mathematical model and a computer simulation have been published (Plaza et al., 1986). These authors specify clearly the dependence of the Raman efficiency on a number of important parameters, namely (i) (ii) (iii) (iv)

Numerical aperture and fiber diameter, Depth of interaction, Interfiber distance, and Angle for nonparallel fibers.

It is also worth noting that other calculations and modelings have been performed by several authors in the case of fluorometric sensors. These calculations, with only minor modifications, can be appUed to Raman systems (Zhong-Yuan Zhu and Yappert, 1992a,b; Moulin et «/., 1993). For example, MouHn et al. (1993) appHed the model of Plaza et al. (1986) to the fluorescence emission. Their results can be simphfied by assuming that the absorption is neghgible, leading to an expression for the fluorescence collection efficiency,

X

y

z

where K characterizes the sample medium, F^. is expressed in sterad mm and W is the beam waist. (a) Single-fiber sensors A single optical fiber can be employed both to transport the excitation laser beam and to collect the scattered Raman radiation. A beamsplitterillumination system is used to couple the input end to both the laser and the spectrometer. The advantage of this configuration lies in the complete overlapping of the excited sample volume and the collection cone. On the other hand, the main drawback to this arrangement is that the Raman spectrum of the fiber material is strongly excited. It is, unfortunately, efficiently collected and transmitted to the spectrometer. The Raman signal of the sample is thus superimposed on an intense background, as shown in Fig. 42, which severely limits the appHcabihty of this sensor, unless the length of the fiber is very short (Walrafen, 1978; Nguyen-Quy Dao et al., 1993; Nguyen Quang Huy, 1993). It is interesting to consider this arrangement from the theoretical point of view. Important calculations have been made that yield values of the depth of sample, and thus the spatial resolution, which are of a useful order of magnitude. In particular, the determination of the effective depth of fluorometric sensors was first made by Deaton (1984). The equivalent

Instrumentation y^

j^

^

y^

^

JJ^

135

T^

> H

;T':

'^ Z w H

" '' \ ;

s

'' -

2 mW :; M

a g

2

w H H

; V



H

< J

S

' ' • ^ - - .

'^

/

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-^

y

"

•

"

'

y

^

1000

2000

3000

WAVENUMBER v (cm"') Figure 42 Stokes-Raman spectrum of a 100-125 |jLm diameter silica optical fiber of 30 m length, at different laser powers.

pathlength was established with the use of an elaborate method by Zhong-Yuan Zhu and Yappert (1992a,b). These results are also of prime importance in the characterization of Raman sensors. Although an optical fiber can integrate the signal coming from an infinite volume, it is of practical interest to determine the contribution of a limited volume close to the end of the fiber. In the present analysis an equivalent volume Feq is defined by a cylinder with a radius equal to that of the fiber core and a length Zgq. The excitation irradiance and the solid angle are assumed to be uniform, and identical to the irradiance and the collection angle at the fiber end. The distance Zeq, called the equivalent pathlength, corresponds to an imaginary cylindrical volume which generates a signal equal to the integrated signal from an infinite sample. From the calculations of Zhong-Yuan Zhu and Yappert (1992a,b), the length of the cylinder is given by 1.076ro

ATTS^ -eq

(T?K)PO^O

~

tana

(84)

where 5x is the total signal collected, PQ is the power of the excitation beam, 17 is the quantum fluorescence yield and K is the absorptivity of the fluorophore.

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ASYMPTOTE

AXIAL DISTANCE Z Figure 43 Integrated signal as a function of sample depth for a single-fiber sensor. For example, with the use of a typical multimode fiber of rg = 60 |xm, tan a ranges in practice from 0.1 to 0.2, so that the equivalent pathlength Zgq is of the order of 300 to 600 |xm. Such a model does not predict the true length from which the signal is collected by an optical fiber sensor immersed in a sample medium. However, it is clear that the dimensions of the imaginary volume are comparable to those of the observed volume in a conventional macro-Raman experiment. In that case the sample medium is illuminated by a lens with a focal length of a few cm and the scattered light is collected by an objective with a moderate //number (tana = 0.2, which corresponds to //2.5, and t a n a = 0.1, which corresponds to //5). It is more important to consider the relationship between the measured Raman signal and the axial distance Z. A complete calculation of the spatial distribution of excitation irradiance and the solid angle of collection enables the integrated signal to be evaluated; thus. S(z)-

d5,(Z),

(85)

which measures the fraction of the total signal generated and collected from a volume of sample contained between the end of the fiber (Z=0) and a plane perpendicular to the axis at a distance Z. The result is plotted in Fig. 43. From the result of this calculation, an effective depth can be defined from which 90% of the signal originates, i.e. Zeff=;^=9Zo. (86) tana This parameter would appear to be much more significant than the equivalent pathlength, since it offers the experimentalist an easy way of fixing a reasonable Umit to the useful thickness of the sample cell in a single-fiber

Instrumentation

Zo =

^^^

137

tana

AXIAL DISTANCE Z

Figure 44 Differential intensity in a single-fiber sensor for a thin layer of sample. arrangement. Again, as an example, with the use of typical values of the parameters for a fiber of diameter Ir^ = 120 |xm, the effective depth which yields 90% of the total measurable signal ranges from 5.4 mm for tan a = 0.1 to 2.7mm for t a n a = 0.2. In order to make a quantitative comparison with the other optical configurations, it is proposed, furthermore, to represent the differential depth of intensity profile, as shown in Fig. 44. The signal intensity I(^z) ni^Y be evaluated theoretically with the use of the same model as before. It can also be measured experimentally from a thin sample layer of thickness dZ, which is perpendicular to the optical axis at a distance Z from the end of the fiber. The intensity 3(ciz) of the Raman signal is maximum for this layer directly in contact with the end of the fiber and exhibits an asymptotic decrease as Z goes to infinity. (b) Double parallel-fiber sensors The use of separate fibers for Raman or fluorimetric sensors has been proposed in order to overcome the drawbacks of the previously described system, essentially by reducing the intensity of the undesirable spectrum of the optical fiber material. For comparison, two different configurations will now be considered, namely (i) Parallel fibers at a distance d and (ii) Convergent fibers at an angle p with respect to the optical axis, as shown in Figs 45 and 46, respectively.

138

M. Delhaye et a/. OVERLAPPING ZONE

EXCITATION

COLLECTION

AXIAL DISTANCE Z Figure 45

Spatial response of a double parallel-fiber sensor.

Instrumentation

•-^ \ in

Z

\ \

z

\

^

>S;^

l / ^ ^ 5

^ ^

1

Ze

H Z H Q

H

< O

AXIAL DISTANCE Z Figure 46

Spatial response of a double convergent-fiber sensor.

139

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M. Delhaye et a/.

For parallel fibers the detailed calculations published by Zhong-Yuan Zhu and Yappert (1992a,b) have been confirmed by experimental measurements with both nonabsorbing and strongly absorbing media. It is concluded that the effective depth Zgff can be given by the semi-empirical formula Z^ff = kAA-^9kBB,

(87)

where A = (rQ-\-d/2)tan~^(a/2) and, as shown in Fig. 45, B = (rQ-\- d/2)tan~^a; k^ and kg are empirical constants (Fig. 45a). Unlike the case of a single-fiber sensor, the excitation and collection cones do not completely overlap in a double- (or multiple-) fiber system. In particular, there is an inactive zone of depth Zj close to the fiber ends, in which the scattering by the molecules excited by the laser radiation is not collected by the fiber. Unfortunately, this inactive zone corresponds to the most efficient part of the sample volume in a single-fiber arrangement. The depth distribution of the differential intensity and the integrated signal curves reflect this drawback, as shown in Fig. 45c. By comparison with the single fiber, the considerable loss of signal due to this inactive zone can be compensated by the absence of a beamsplitter and, furthermore, by the use of multiple collection fibers. (c) Nonparallel fiber sensors A significant improvement in the depth discrimination is obtained by replacing the parallel fiber by convergent fibers (double or multiple). As shown in Fig. 46, this configuration shortens the inactive zone and produces a maximum signal at the distance Z ^ , where the cones of excitation and collection completely overlap. However, the useful depth of sample is limited to the distance (Zg — Zi) at which the sections of the cones are tangential. This property may be advantageous in practical experiments where it is desirable to avoid spurious signals from the back of the sample cell. Another advantage in quantitative analysis is that the useful volume of sample is limited geometrically and held constant, thus eliminating some of the causes of error. 3. Conclusion Some significant features of direct-coupled fiber optrodes can be derived from these models. They are in general confirmed by experimental measurements, namely: (i) The monofiber optrode is the simplest and most successful configuration, because it provides perfect overlap between the excitation and collection fields at the fiber end. Unfortunately, a considerable drawback is the

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141

presence of the extremely intense spectrum of the fiber itself, so that the detection of analytical Raman signals is possible only if very short fibers are employed. (ii) For double or multiple fibers, the Raman spectrum of the return fiber is also often a severe limitation when a sample reflects a significant part of the excitation beam. This effect limits the applicabihty of this arrangement to gases or neat liquids. (iii) The collection efficiency is strongly dependent on the distance between the collection and excitation fibers. (iv) As would be expected, best results are obtained for an optrode with large numerical aperture collection fibers. (v) Decreasing the diameter of the excitation fiber leads to a significant increase in irradiance at the sample medium and consequently improves the efficiency; at the limit a monomode excitation fiber would be optimum. However, problems then arise at the laser-to-fiber injection, where the maximum acceptable beam power and the injection losses are the limiting factors. (vi) As for the spatial resolution and microprobing capabilities, it is found that the single-fiber optrode is the only one which defines a microsized sample area whose dimensions are comparable to the core diameter. The lateral resolution hes in the range 5-10 |xm for a monomode fiber. However, the depth discrimination is much less than for a conventional Raman microscope. A possible future improvement in the performance of optical fiber systems could come from the technique of near-field microscopy, which provides a sub-wavelength resolution and nanometer depth discrimination. However, at present the applications of this fascinating development are limited to those spectroscopies where intense signals are available.

D. Indirect-coupled Raman Sensors As recalled above, the direct coupling of the optical fiber ends to the sample medium introduces severe limitations in micro-Raman appUcations, namely, (i) Strong interference of the background spectrum due to the fiber medium, and (ii) Poor spatial resolution. To overcome these difficulties, it has been suggested (Da Silva et al., 1994; DILOR, 1994) that an ancillary optical system is used to obtain optimized coupling of the optical fiber to the sample. Indirect coupling improves the performance in two respects. First, the

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L-

F^

LASER EXCITATION FIBER

SAMPLE

RAMAN RETURN FIBER Figure 47 Principle of the indirect-coupled measuring head with two spectral filters; Fii narrow-band interference filter, F2: notch rejection filter.

interference due to the Raman spectrum of the fibers can be eliminated by means of spectral filters placed between the fiber ends and the sample. Second, matching the fiber ends to the sample by means of objectives results in clearly defined lateral and axial spatial resolutions which are comparable to those of conventional Raman microprobes. Furthermore, some advantages result from the optical separation. All ancillary optics can be contained in an enclosed measuring head which protects the optical fibers from corrosion or contamination. A non-contact probe is more versatile for many analytical applications, as it permits the introduction of protective windows, which are especially useful when the sample is toxic, corrosive, radioactive or under severe physical conditions of pressure or temperature. A schematic layout of an indirect-coupled measuring head is shown in Fig. 47. Two separate optical fibers are employed; one is for the laserbeam excitation and the other for the Raman light which returns to the spectrometer. In principle each of these fibers has to be optically conjugated

Instrumentation

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to the sample via convergent lenses or mirror systems whose aperture is correctly matched to the acceptance cones of the fibers. Two separate spectral filters are usually necessary to eliminate the Raman (or luminescence) spectrum originating from the fiber material; they are: (i) A narrow-band interference filter, which is centered on the laser wavelength. It is placed on the light path between the excitation fiber end and the sample. This filter efficiently blocks the intense side-bands due to the Stokes- and anti-Stokes-Raman scattering by the optical fiber. (ii) A notch rejection filter, which is placed in the pathway of the scattered radiation collected from the sample in such a way that the return fiber is protected against the intense, diffuse, backreflection of the laser radiation onto the sample. The interference or holographic filters must be employed in a parallel beam, with an accurate adjustment of the angle of incidence. Thus, the optimal optical system, as shown in Fig. 47, comprises at least two convergent elements Li,L2 and L3,L4 in each beam - excitation or collection. In this configuration the angle between the excitation and the collection beam, as well as the sohd angle of collection ft, may be adapted to specific requirements. However, for most analytical applications it is preferable to combine the excitation and collection beams with the use of a single objective O^i in a backscattering arrangement. This configuration is shown in Fig. 48. The only drawback is then the loss of both incident power and Raman signal due to the beamsplitter. A further improvement can be obtained by employing a dichroic beamspHtter, which may, under certain conditions, play the role of the notch filter F2 in Fig. 48. This optical configuration, as shown in Fig. 49, usually makes use of a holographic notch filter, working by reflection to direct the excitation beam towards the sample, and by transmission to pass the Raman-scattered light. As explained in Section IV of this chapter, such an arrangement benefits from the complementary spectral responses of an interferometric or holographic filter in transmission and reflection (see Fig. 35). With the elimination of the undesirable spectra of the fibers, these measuring heads enable the analysis to be made of any kind of sample - not only clear Hquids, but also turbid suspensions of scattering particles, and even highly reflective solids such as amorphous or polycrystalline powders. The spectral range of the system described above is determined by the response of the notch rejection filter, which usually covers the middle range, from 150 to 3500 cm~^. A progressive attenuation of the low-frequency domain does not constitute a serious Umitation for most analytical remote measurements. To improve the versatility of the system it is advantageous to provide the measuring head with interchangeable front objectives O^, By a proper choice of this objective, an optimized coupling of the optical fibers

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LASER EXCITATION FIBER

RAMAN RETURN FIBER

SAMPLE

Figure 48 Optical-fiber, indirect-coupled sensor with collinear excitation, backscattering and spectral filtering to reject the spectrum of the fiber.

RAMAN RETURN FIBER

LASER EXCITATION HBER

i

MIRROR

Figure 49 Principle of a Raman optical fiber probe with a dichroic, holographic beamsplitter DBS (DILOR Superhead).

to any specific sample is made possible, resulting in significant advantages, namely (i) Maximum irradiance of the excitation laser beam in the secondary laser beam waist formed in the observed sample volume,

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(ii) Quasi-constant, wide solid angles of scattered light collection. (iii) Concentration of the whole signal originating from an individual experimental site in a single return fiber. (iv) A high intensity of Raman signal is obtained from a short depth of sample, easily controlled by the choice of the front objective. Thus, quantitative analytical measurements are not perturbed by any variation in radiation due to the cell walls. (v) If needed, the interchangeable front objectives enable adaptation of the available working distance. As wide collection angles are desirable, the best spatial resolution and efficiency are obtained when short focal length objectives are employed. However, when a large amount of sample is available the use of long working distances (cm to dm) may compensate for the reduction in the soHd angle and local irradiation by an increased effective depth. (vi) The working distance can be chosen by taking account of the available sample volume and shape, together with the necessary protective windows when the conditions of measurements require high temperature, controlled atmosphere or high pressure. From the point of view of spatial resolution these optical coupling systems meet the conditions required for a confocal microscope. Their performance is comparable with that of a conventional instrument in macro- or microRaman spectroscopy. It is important to note, however, that significant differences appear in the order of magnitude of the optical parameters. In a real confocal microscope, which uses small-diameter pinholes, spatial filters and high magnification factors, the performance is essentially limited by diffraction. On the other hand, for most analytical applications of remote, optical fiber devices, multimode fibers are employed with core diameters of 50-200 |uim and low magnification factors of the ancillary optical system. Under these restrictive conditions, the spatial resolution is no longer limited by diffraction, so that a valuable approximation is obtained by simply applying geometrical optics. For these reasons, it would seem to be preferable to call such devices 'pseudo-confocal' or 'confocal-like' systems. Consider, for example, a retroscattering measuring head in which the excitation and collection beams are collinear, as shown in Figs 48 and 49. The apertures of the lenses Li and L4 are fixed by the characteristics of the optical fibers, so as to obtain matching with their acceptance cones. Typically, with N. A. = 0.2 and the choice of a lens Lj of focal length / i = 15 mm, the diameter of the parallel beam in the filter section is nearly 6 mm. The lateral resolution and depth discrimination of this kind of Raman sensor are determined by two pairs of parameters: (i) The focal length / ^ and the effective numerical aperture N.A.m of the front objective Om-

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M.Delhaye et al.

(ii) The core diameters of the excitation fiber Dex and of the Raman return fiber DRam. Usually, when maximum spatial resolution is not required, two identical fibers are employed so that D^^ = Z^Ram and the focal lengths /i and f^ are equal. Both fiber ends are then imaged onto the sample with the same magnification factor in order to maximize the intersection of the excitation and collection beams at the sample. The lateral resolution can be simply defined approximately by the beam diameter at sample D^

D, = D,P-^\.

(88)

The axial resolution is usually derived from the differential depth profile of intensity, which is experimentally measured by translating a thin layer of sample perpendicular to the optical axis and plotting the relative signal I{dZ) versus the axial distance Z. A typical curve of the differential intensity profile is given in Fig. 50. In practice, the depth of focus is often defined by the full width at half maximum (FWHM) of these curves. An approximate evaluation of the depth of focus is given by

FWHM = V2 {-^^

- J^^,

(89)

\tana/ N.A.^ where a is the semi-angle of aperture and N.A.^ the effective numerical aperture of the front objective O^. For a given measuring head, where the fiber-core diameters and the focal lengths /i and f^ are fixed, the depth of focus may be varied as a function of/m/N.A.m, the ratio of focal length to numerical aperture of the interchangeable front objective. Table 2 gives some typical values for a DILOR Superhead with 100 |xm fibers and fi=U = 16 mm. As expected, by comparison with a conventional microscope, the Raman signal intensity is proportional to (N.A.j^)^ when a thin sample is under observation. The response is more complicated for a thick, transparent sample medium. In effect, there is a compensation of two opposite effects, when the focal length of the front objective is varied. If the focal length increases, the sample irradiance diminishes, while the effective depth of sample increases. As a result, a maximum intensity of the Raman spectrum is observed when the magnification factor/m//i approaches unity. Then, the lateral resolution becomes equal to the fiber-core diameter. Instead of using two identical, large-core fibers, it may be interesting for some applications to improve the spatial resolution and to increase the irradiance of excitation in the sample by employing an excitation fiber of smaller diameter. At the limit, the use of a monomode excitation fiber.

Instrumentation LASER EXCITATION FTOER

RAMAN RETURN FIBER

AXIAL DISTANCE Z Figure 50

Spatial response of an indirect-coupled Raman sensor.

147

148 Table 2

M.Delhaye et a I. Typical values of FWHM for a DILOR Superhead with 100 jxm fibers and / i = / 4 = 16 mm.

Objective focal length / ^ (mm)

N.A.^

FWHM (mm)

3.5 8 40

0.5 0.35 0.07

0.06 0.2 5.5

together with a short focal length, high-aperture front objective results in a diffraction-limited laser-excitation waist which provides the lateral resolution of a classical microprobe. In order to obtain convenient and stable alignment of the ancillary optics, it is advantageous to employ a multimode fiber with a large core diameter at the output of the sensor to transfer the Raman radiation to the spectral analyzer.

E. Conclusions

It is meaningful to compare the results of the above analysis with the spatial properties of direct-coupled, optical fiber sensors. It is clear that the principle of indirect coupling, with the aid of focusing lenses and filtering systems, provides a much better spatial resolution than can be obtained with the direct-coupled configuration. In addition, the capability of optimizing the lateral and axial resolutions by means of interchangeable objectives provides the sensor with a decisive advantage for Raman microprobing, and thus in situ local analysis. Indirect coupling also provides the optical fiber probe with efficient spectral filtering, resulting in a dramatic improvement in the analytical capabilities of the system by virtually eliminating the interference due to the spectrum of the fibers. This unique advantage is demonstrated in Fig. 51, which presents a comparison of the spectrum obtained with a length of fiber direct-coupled to the sample and that recorded with the use of a DILOR measurement head incorporating two complementary indirect-coupled fibers. Some other examples of applications of this system are shown in Figs 52 and 53. Finally, it is apparent that remote micro-Raman analysis by means of optimized optical fiber sensors offers many advantages when the sample has to be observed under hostile or hazardous environmental conditions. In situ measurements with the use of several hundred meters of optical fibers are possible without sacrificing most of the spectral and spatial advantages of the Raman microprobe technique. The same kind of instrument may also prove to be useful when equipped with short-length fibers and versatile accessories for the in situ analysis of objets d'art, as well as in forensic investigations (see Chapter 10).

Instrumentation

(a)

149

WAVENUMBER (cm"')

H

g a 2 w

H H <

-J

(b)

WAVENUMBER (cm"')

Figure 51 Comparison of the Raman spectra of a white pellet of aspirin, (a) Direct-coupled optrode showing the hmitation in the 200-1000 cm~^ region due to the silica spectrum of the optical fibers; (b) Indirect coupHng system (DILOR Superhead) with a double spectral filtering system which permits detection of the aspirin bands (A-H) by suppression of the spectrum of the fiber.

150

M.Delhaye et al.

3500

2500

1500

WAVENUMBER (cm-^)

> H

(b)

1

s o 2

w

H

 <

L._ i

3400

J

.,

3000

1600

1200

800

WAVENUMBER (cm"') Figure 52 Raman spectra of a polycrystalline powdered, white sample of imidazole obtained with an indirect-sampling, optical fiber system (DILOR Superhead) with an Induran multichannel spectrometer; fiber length 100 m, excitation at 514.5 nm. (a) Laser power 3.5 mW, recording time 1 s; medium dispersion with a 600 Hnesmm"^ grating, (b) and (c) Laser power 7mW, recording time I s ; high dispersion with an 1800 lines mm~^ grating.

Instrumentation

3500 (a)

2500

500

WAVENUMBER (cm"^)

1200 (b)

1500

151

800

WAVENUMBER (cm ')

Figure 53 Raman spectra of polycrystalline graphite obtained with the same optical system as in Fig. 52. (a) Laser power 14.5 mW, recording time 5 s; 600 Unes mm~^ grating, (b) Laser power 16 mW, recording time 20 s; 1800 lines mm~^ grating.

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VIII. A BRIEF SURVEY OF DIGITAL SIGNAL PROCESSING A. Introduction The appearance of this section in a book completely devoted to Raman microscopy may surprise some readers. However, in the spectrometers now employed in this area (classical scanning, multichannel or Fourier transform instruments), monitoring, data acquisition and processing are performed numerically by means of microcomputers. Moreover, as will be seen in the presentation of the various applications in the following chapters, analyses of either microsamples or macrosamples with the use of a Raman microspectrometer often lead to very weak spectra, blurred by noise or perturbed by stray light emission (e.g. fluorescence). In order to be readable, these spectra usually require efficient signal-to-noise-ratio (S/N) enhancement or specific data treatment processes which are easily performed by simple numerical methods. Although it is accepted that improperly used spectrometers may yield incorrect spectra, it is not generally understood that inappropriate digital signal processing methods may also yield erroneous and misleading data. In order to appreciate the benefits afforded by computerized data, as well as the use of numerical methods (and possibly the improvement of commercially available ones), it is necessary to understand the relevant mathematics. In particular, the spectroscopist must be in a position to evaluate the limitations and avoid the pitfalls commonly encountered in digital signal processing. In the following paragraphs a brief survey of basic digital signal processing methods is presented. This summary includes certain concepts of the sampling of analog data, the traditional methods of improving the S/N (such as signal averaging, filtering, windowing and smoothing), as well as some practical applications to Raman spectroscopy. However, prior to these topics, the fundamental mathematical tools such as the d or impulse function, the convolution theorem and, in particular, the Fourier transform - the key operation in signal analysis and processing - must be introduced.

B. Mathematical Background 1. The Impulse and Sampling Functions As an introduction to the impulse d function (Dirac), consider the sampUng process in which a continuous analog signal s(v), here the detector output voltage 5^ as a function of wavenumber, is converted into discrete ampUtudes at specified sampling intervals, AT' (cm~^). Physically, this operation can be represented by a switch which opens every ^v wavenumbers for a short interval rcm"^ (Fig. 54a). The sampling process thus produces a train of

Instrumentation

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/(V,T)

'I (a)

-Av

0

Av

3Av

5Av

7Av

s(v) (b)

WAVENUMBER (cm"')

.^(v)

\

(c)

/ >

A

\-A

L 0 Av

3Av

H'-T5Av

7Av

Figure 54 Sampling process, (a) Switching function, (b) Original analog Raman signal, (c) Sampled Raman signal.

narrow pulses, f(v, r) that can be treated as Dirac impulses, 8(v), in the limit that T«Av. From this point of view, the 8 function can be expressed in the form d(v)=

\imf(v,T).

(90)

Usually the 8 function is defined with the use of the classical variable, t (Brigham, 1974) as ^(^-^o) = 0 if

t^to

(91)

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M. Melhaye et al.

and [^

d{t-tQ)dt=l,

(92)

J — CO

with the very important property that for a given function x{i) oo

8{t-to)x(t)dt

= x(to)

(93)

The ideal sampHng operator consists of an infinite series of 8 functions, sometimes called the 'Dirac comb', or 'shah' (Hebrew J_u). This function (Chamberlain, 1979; Jansson, 1984; de Coulon, 1984) can be defined by

^(^)= Av

2

8{v-nM^),

(94)

n = — oc

where n is an integer. Then, returning to the samphng problem, the sampled signal of s(v), namely s(nAv), is now expressed by Sp{nv) = iu(v)s{v),

(95)

as shown in Fig. 54. With the use of the definition and properties of the sampling operator Eq. (95) can be written in the form n = + oc

Sp{np)=

2

s{nAV)8(v-nAv).

(96)

This equation is the mathematical representation of the sampling operation, whose significance will be discussed in the following paragraphs. 2.

Convolution

The concept of convolution is very familiar to spectroscopists. Moreover, their daily experiences abound with phenomena that can be represented by the mathematical operation of convolution, as it describes the process which causes 'blurring' or spreading of a physical observation. Consider the classical example (for the spectroscopist) of the effect of a nonzero slit width on a spectrum. The lineshape recorded in an observed spectrum is the convolution of the 'true' (physical) lineshape with the slit function of the spectrometer. As is well known, this effect often limits the resolution of the instrument. This example will be treated in detail in Section D. The convolution operation (noted by *) of two functions e(t) and h(t) is defined by e(t) * h(t) =\

e(e) h(t -e)dd

(97)

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155

where ^ is a dummy variable. This operation is both (i) commutative and (ii) associative. Furthermore, (iii) the d function is its unit element. Thus with the use of (i) along with the properties of 3 (Eq. 93), the appHcation of (iii) to any function h{i) leads to d{t)^h{t) = h{t).

(98)

This equation can be written in integral form as h(e)d(t-d)dd

= h{t).

(99)

)

In an experimental situation where Eq. (99) can be applied, the impulse response, h(t), can be considered to be characteristic of the system. This function is also known as the apparatus (or instrument) function, which must be determined in order to perform a deconvolution. This operation allows the 'true' spectrum, as in the example given above, to be recovered. From Eq. (99) the convolution also allows the translation operation of a function f(t) to be defined, namely f(t-to)=m*d(t-to)

(100)

This important property is very useful for handling sampled data, as demonstrated in the following section. 3. The Fourier Transform So far the sampHng process has been described as a formal operation; this discussion has provided an opportunity to introduce certain mathematical techniques. However, if it is desired to recover perfectly the signal s(v) from the set of numbers s(nAV) [see Eq. (96) and Fig. 54], it is obvious that some considerations regarding the sampling interval, Av, must be specified. To establish these criteria (see the discussion of the sampling theorem. Section VIII.C.2), the knowledge of the frequency content of the signal, i.e. its spectrum,* is necessary. This spectral analysis can be performed with the use of the Fourier transform (FT). For this reason the concept of the Fourier transform will now be introduced and some of its most useful properties will be summarized. The Fourier transform F(v) of a signal f(u) is given by r +00

F{v)=

/(i^)e-2^^"dw.

(101)

*The present work is largely devoted to Raman spectroscopy. Thus, the term 'spectrum' is understood to mean an array of one-dimensional data, i.e. the scattered light signal (volts, cps, etc.) versus wavenumber. Confusion sometimes arises when the Fourier transform of this array is discussed, as it is often referred to as a spectrum. The latter array will be referred to here as the 'Fourier spectrum'.

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M. Melhaye et al. f{u)

-^

-^F(v) p sine (V p)

n (M/p)

0

-p/2

p/2

A (w/p)

0

1/p

Xn{v)

;\iMu) l/Av

A

ttiiitttiii.. -^\'

^\'

0

la

0

A

A

IMv

alln

Figure 55 Some important Fourier transform pairs, (a) Boxcar or rectangle (| |) and sine cardinal (sine); (b) triangle (A) and sinc^; (c) Dirac comb or shah (I I i^; (d) exponential e~«l"l and Lorentzian lala^ + (lirvf-', (e) Gaussian; e~'^^ ;

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In this expression u is an independent variable of the measurement space (wavelength A, wavenumber v, time t) and v the corresponding 'frequency', in the Fourier space (A~^,x, v). On the other hand, in the case where the frequency function F{v) is known, f{u) can be retrieved by making use of the inverse Fourier transform f{u)=[

"F(u)e-2^^"dz/.

(102)

The functions F{v) and/(w) are called Fourier transform pairs. Some of the most important Fourier pairs in spectrometry and digital data handling will now be presented without any derivation. The examples illustrated in Fig. 55 are the rectangle or boxcar (| |) and sine (sine cardinal), triangle (A) and sine squared (sinc^), Dirac comb ( Mi) and the two famihar Gaussian and Lorentzian peak-Hke functions. Readers are strongly urged to perform some of these computations themselves with the use of the obvious symmetry properties of these real, even functions, in order to get a Httle practice in handHng FT pairs. For more details, the excellent books of Bracewell (1978), Brigham (1974) and Chamberlain (1979) can be consulted. It should be noted that these integrations [Eqs (101) and (102)] are very efficiently carried out on a microcomputer. The fast FT (FFT) algorithm (Cooley and Tukey, 1965; Brigham, 1974) can be employed to reduce the calculation time if the number of data points is equal to an integral power of two. The convolution theorem, which is fundamental for understanding the problem data acquisition and, thus, the performance of spectrometers, will now be presented. The theorem states that the convolution of two functions in u space is mathematically equivalent to the product of their transforms in V space and vice versa. Thus, if s{u) = e{u)^h{u)

(103)

S{v) = E{v)'H{v).

(104)

then In the FT pair notation, Eqs (103) and (104) can be written as e{u) * h{u) ^ ^ E{v)' H(v)

(105)

e{u)' h(u) ^ ^ E(v) * H(v).

(106)

and An example of the application of the convolution theorem is provided by the Fourier transform pair given in Fig. 55b. By graphical construction (Marshall, 1982), it is easy to verify that

n(^)*rn(")=A(..)

(io7)

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and that the convolution theorem [Eqs (103) and (104)] leads immediately to the relation A(w)^^sinc2(u).

(108)

The great importance of this theorem in various branches of spectroscopy, as well as more generally in signal analysis and data processing, will become apparent in the following sections.

C. Measurement and Sampling of Analog Signals 1. Generalities and Initial Hypotheses Consider again the signal, s{v), from a Raman detector, as introduced in Section VIII.B.l, and assume that its Fourier spectrum, 5(jc), is band-limited to Bern (Fig. 56). The samphng of s(v), as expressed by Eq. (95), is represented by the modulation of the analog signal by the sampUng operator I I iAr(^)- defined by Eq. (94). The role of the sampling operation is to transform the analog signal, 5(1^), into a set of equally spaced values, ^(nAP), without any loss of information. A successful sampling operation, which is determined by a correct choice of the sampling step, Az^, allows an unambiguous (perfect) original signal to be reconstructed from the sampled values. This operation can be achieved if, and only if, the requirements of the sampling theorem are fulfilled. This theorem states that in the case of a band-limited signal such as siv), it can be 'perfectly' reconstructed from the samples s{n^v) provided that the samphng rate, 1/AI^, is greater than twice its highest frequency, B, that is 1/Az^>25. The mathematical proof, as well as some illustrations and consequences of the practical appHcation of this theorem, will now be presented.

2. Applications of the Sampling

Theorem

Consider the signal s{v) represented in Fig. 56a, and S{x), its Fourier spectrum (Fig. 56b). The Fourier spectrum is a band-limited function in that S{x) = 0,

for

\x\^B.

(109)

The sampling of s{v) leads to Spiv) as given by Eq. (96), Sp{V) = s{v)'^{v).

(110)

With the appUcation of the convolution theorem [Eqs (103) and (104)] this operation appears in the Fourier domain as the convolution product of S{x)

Instrumentation 5(V)

(b)

159

I S (x)

-B (c)

I I I I I II

^(v)

rUJK)

(d)

1

I

'

1 ....

.... -1/Av

-AV (e)

11 f ft I

j(v)^(v) 1

"

5(A:)*^(X)

f1 t f I Av1 ^ ^ K i

1/Av

'

i 1

-1/Av j

j-l/Av

-1/2AV 1/2AV

(g)

5(V)^U(V)

^ttflftk .11^1 fl tffttttfi 1 1 1 1 1 1 1 litft1 1 ^1K-Av

5(x)*^(;c)

(h)

ff 1

1/Av

1/Av

1

-1/2AV

1/2AV

H{x) (i)

Av

-1/2AV

1/2AV

H{x).[S{x)^;d}{x)

(J)

-B

B

Figure 56 Schematic representation of the various steps in the sampling process; note that (e) and (f) illustrate the origin of aliasing (see text). The recovery of the analog signal from samples is depicted in parts (h)-(k).

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and {II^v) \ I \(x). [Note that the Dirac comb I i i is its own Fourier transform (Fig. 55c).] +00

S{n^x) = S{x)*{\l^l})

2 ^ 3{x-nlM^)

(111)

and, with the use of Eq. (100), +00

S{n^x) = {\I^V) 2 ] S{x-nlM}).

{Ill)

/7=—oc

It can be seen from Eq. (112) that the sampHng of s{v) produces in the Fourier domain a periodic repHcation of S{x) at every interval \l^v. The consequences of this operation are illustrated in Figs 56f and 56h for two different values of iiv. In the case where AP is small enough (Fig. 56g), it appears that S{n^) is equal to S{x) within a constant 1/Az^ in the interval [-l/(2Ai7), + 1/(2AI^)] (Fig. 56h). The quantity 1/(2A7^) is called the Nyquist frequency. The endless replication of the signal, as suggested in Fig. 56f, is usually referred to as 'aliasing'. However, from Fig. 56f it can be seen that when ^v is chosen too coarsely (Fig. 56e), the replicated Fourier spectra overlap on both sides of points x^ = n/(2Ap). In the latter case S{x) cannot be recovered from S(nAx) and obviously s(p) cannot be reconstructed from the set of numbers s(nA'P). This example can serve as a demonstration of the sampHng theorem, which is restated as follows: In order to recover perfectly s{v) from s{nAv), the signal must be sampled with Av^l/2B, where B is the highest frequency in its Fourier spectrum. If this criterion is satisfied, a general formula for the reconstruction of s{v) from s(nAV) can be derived. Recall that in the present example S{nAx) is equal, within a constant 1/Av, to S(x) in the range [-1/(2A7^), + 1/(2AP)] (Fig. 56h). Then, for all x, S(x) = H{x)'S{nAx).

(113)

This relation expresses the filtering of S{nAx) by H(x), the transfer function of the boxcar filter, whose characteristics are given in Fig. 56i. If the inverse Fourier transform is taken and the convolution is applied, Eq. (113) can be written in wavenumber space as s(v) = hiv)*Sp{V),

(114)

sin(7ri^/A^) "^^^^ = (.WAP) •

^^^^^

where

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Finally, with the use of Eq. (96) and the definition of the convolution product [Eq. (99)], Eq. (113) becomes /-x s(v)=

x^ / A-x sinf(7r/A7^)(I^-nAP)] > s(n^v)—/\, , ; ^ T-r^' „^^ (77/A]^)(7^-AzAz/)

.^_. (116) ^ ^

The sum in Eq. (116) is Whittakers cardinal function (Stearns, 1975), which allows the reconstruction of s{v) for all v from a knowledge of only the samples s{n^v). This result is valid as long as siv) is band-limited to B cm, with B^ll{2M}). 3. Sampling of Analog Signals In practice the above condition is never fulfilled, since experimental analog signals are not simply band-limited (due to their finite duration, noise, etc.); furthermore, aUasing appears. The solution to this problem consists of filtering the signal before the sampUng process with an appropriate low-pass filter. This procedure is called analog presampling filtering (APF). Thus, the APF process band-limits the input signal, as required by the sampUng theorem. The sampling rate, which is determined by the conditions imposed by the sampUng theorem, is then calculated from the cutoff frequency characteristics of the filter. With the attenuation of the high-frequency content of the signals before they reach the sampler, such filters largely suppress the effects of the aliasing process; for this reason they are called 'antiaUasing filters'. The reader is referred to application textbooks (Williams and Taylor, 1985; Strassberg, 1991) for information on how to construct antiaUasing filters. In the foUowing paragraphs some comments wiU be made and illustrations presented of the sampling process. Specific examples from optical spectroscopy, and in particular Raman spectroscopy, wiU be discussed.

D. Practical Considerations in the Digitizing of Raman Spectra 1. Slit Function and Line Shape In the description that has just been given of the sampling theorem, the Nyquist frequency, the concept of band-limited signals and Whittaker's reconstruction from the sample set have all been introduced in terms of mathematical functions rather than physical signals. In the following section a detailed analysis will be made of the sampUng process of Raman bands which contribute to a compUcated spectrum as recorded by a classical, scanning spectrometer. For simpUcity, it will be assumed that the signal (spectrum) to be sampled is noise-free, as if the APF process were very efficient.

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Consider a simplified single-stage, scanning Raman spectrometer. It includes, essentially, a highly dispersive monochromator and a very sensitive detector, such as a photomultiplier (PM). The PM output signal, s{v), is fed into a microcomputer through an analog-to-digital converter (ADC). Any accurate numerical analysis of the spectrum, i.e. an estimation of the position, height and width of each Raman band or line, requires that the spectrum be correctly digitized. The major problem for this class of signal is that it is not perfectly band-limited to ^ cm. To obey the sampling theorem an upper 'frequency' limit must be defined above which S{x) is very small. In practice this condition implies that a reasonably good approximation to the band-limit B can be found. Two classes of Raman spectral feature will now be examined. First, consider a Raman spectrum which consists of very narrow lines, and second, a spectrum of broad bands with Lorentzian shapes. Because the spectral sUt of the monochromator has a nonzero width, /(cm~^), the output spectrum, s{v), will in the first case consist of features which appear to be broader than in the true spectrum. In the second case, however, the bands preserve approximately their correct shapes. Indeed, if l^via is the Raman hnewidth (FWHM), then for narrow bands Avi/2«f, whereas for broad bands it will be assumed that AI^i/2>5/. In the former case the shape of a Raman line will be imposed by the instrumental profile of the monochromator function. As is well known, when the entrance and exit slits of a monochromator are of equal width, the instrumental function is given by the convolution product of the two rectangular sHt functions Ai{v) and A2(i^). This consideration leads to a triangular function, g{v) [see Eq.(107)], whose characteristics are given in Fig. 57a (Bousquet, 1969). The observed broadening effect is again the result of a convolution process between the true input spectrum and the instrumental function. Thus, in the case of very narrow Raman fines, which can be approximated by 8 impulses, the output spectrum s{v) will appear as a collection of wider fines with triangular profiles, since siV)^^S(V-Vd*g{V)

(117)

i

and hence, s(V)^^g(l}-Vi)-

(118)

2. The Sampling Steps as a Function of Spectral Lineshape and Width (a) Raman lines with triangular shapes To find the band-limit B, the Fourier transform of the lineshape function, gij}) must be carried out. This transform, G(jc), has already been given in

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163

g(v) = Ai*A2 A,(v) (a)

Aiiy)

h

f I

f

ENTRANCE SLIT

EXIT SLIT

FT Amplitude

(%) (b)

1

4.5

2

1.6

3

0.8

4

0.5

5

0.3

G(x)

Figures? (a) Transfer function^g(i^), of a single-stage, scanning spectrometer, (b) Fourier transform, G{x), oi g{v)\ the relative amplitudes of the secondary maxima are given in the table (see text for explanation). Fig. 55b and Eq. (108). In the present case it can be written in the form , sin^(7r/x) -={hffsmc^{TTfx).

G{x) = {hff

(119)

This function, which is shown in Fig. 57b, has a principal maximum at x = 0 ; the minima are found at X = klf

for

/:(integer) > 1

(120)

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M.Delhaye et a I.

and secondary maxima at Xk = {2k + \)l{lf),

(121)

with the corresponding ampHtudes Ai^ given by Ak = A{hffl[7r'{2k+\f].

(122)

The relative amplitudes (A/^/h^f^) versus k are given in the table of Fig. 57b. It appears immediately that for k>4, G{x) is very small {Ai^lh^f- < 0.5 X 10~^) and it can be assumed that the corresponding minimum, 4//, is a good approximation to the band-limit B. Then, with B = 4/f, the triangular Raman lines g{v) of width / can be correctly reconstructed from samples taken every//8cm~^ (b) Broad Raman bands with Lorentzian shapes The Raman spectrum is now constituted of broad Raman bands, L{v), whose widths, A7^i/2, are much greater than the spectral slit width / of the spectrometer, i.e. Ai^i/2>5/. In this case the observed spectrum s(v) can be represented by s{v) = ^L{v-v^*g{v),

(123)

which will appear as the Raman spectrum 5(i7)=.2;L(^-I7,).

(124)

The Fourier transform pairs, Hv) and /(x), are given in Fig. 58 (see also Fig. 55d). If it is assumed that l{x) is complete at x = 10/a, [/(10/a)//(0)< 10""^], the signal L{v) can be correctly recovered from the values sampled at a rate l/Ai^>20/a, i.e. a sampling step Lv^ 7rAZ'i/2/20 ~ A^'i/2/6. This condition is, however, much stricter than that stated above in the case of triangular lineshapes. It is generally sufficient to take B = 5/a, or [/(5a)//(0)]<0.7x 10"^, which leads to M^^AVy2/3. For broad, smooth band profiles (Lorentzian, Gaussian, etc.) this rule, i.e. three points per bandwidth (FWHM), is commonly accepted. In summary, the instrumental function of a monochromator can be reconstructed correctly from sampled values with the use of at least eight points per spectral slit width. This principle is of great importance in numerical deconvolution in which the apparatus function is needed with great accuracy. On the other hand, as a rule of thumb, at least three points per linewidth (FWHM) are necessary to define the profile of a Raman line correctly. These rules may apply approximately for multichannel

Instrumentation

EXPONENTIAL

165

LORENTZ

Figure 58 Typical Lorentzian Raman line, L{v) = a^la^ + Aip-i?, and its Fourier transform, l{x) = ^ae""'^'; Ai^i/2 is the full width at half maximum (FWHM).

Spectrometers, which employ linear, diode-array detectors (see Section V). In this case the word 'points' must simply be replaced by 'diode output'. Once the spectra have been digitized, numerical methods can be used to improve the S/N. These methods include signal averaging, filtering, windowing, or apodization, and smoothing. These topics will be developed in the last part of this section.

E. Digital Data Methods for Improving the Signal-to-Noise Ratio Although some digital processing methods have been known to chemists for many years (Savitzky and Golay, 1964), it is only comparatively recently that these methods have become important. This development is due to the general availability of modern computerized spectrometers. Thus, signal averaging, filtering, windowing and smoothing are commonly implemented methods which, when properly used, lead to significant improvement in spectral quality. In addition, specific computer manipulations such as baseline correction, polynomial fitting, band decomposition ('deconvolution') and interpolation are often included in commercial spectrometer software packages. Some of these methods will be examined below in an attempt to outUne their main characteristics. All users of these 'numerical recipes' should have at least a basic understanding of how they work in order to avoid their misuse and classical pitfalls. i. Signal Averaging The technique of signal averaging has been known for a long time and it has been described in a number of excellent papers. In this method successive identical signals are added m times so that the S/N is enhanced by a factor of V m . In his review article Ernst (1965) presents the mathematical basis

166

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for the evaluation of this method, especially in comparison with signal measurements which require the same total time, Ar, necessary to record the m accumulated signals. Consider first the mechanism of this V m improvement. After the accumulation of m successive, identical scans of duration TJ, the signal, which is the detected Raman spectrum, will increase by a factor of m; then, s^(j}) = ms{V).

(125)

The noise, on the other hand, is a random process with an average value of zero. It is characterized by its RMS value, which is obtained from the noise power, PN- Thus, RMSnoise = < ' .

(126)

After averaging m signals the total noise power P{^ is found to be equal to P{^ = mP^.

(127)

With m accumulations S/N is, from Eqs (125)-(127), given by S/N = ms{p)/VmP^^^.

(128)

Equation (128) is an expression of the V m law. It should be noted that this law is applicable in many situations, even if the noise is complicated by slow drifts and/or large low-frequency contributions (Aubard, 1980). A second important point, of particular interest to Raman spectroscopists, concerns the advantage of the signal-averaging method over the use of a single measurement obtained in the same total time, A/. In principle, these two methods lead to the same improvement in S/N, V m and V ( A / / T I ) , respectively. However, in practice it has been demonstrated that in modern multichannel Raman spectrometers which employ cooled detectors, especially CCDs, a single sweep lasting At yields a better S/N.

2. Filtering and Windowing In the broad sense, filtering is a process commonly used to reduce or eliminate parts of the Fourier spectrum of a given signal. Filters are most commonly used to remove high-frequency fluctuations (noise) from the low-frequency part (the signal); such filters are called low-pass filters. However, it is also possible to construct high-pass filters that eUminate slowly varying signal drifts such as the fluorescence background in the resonance Raman spectra of dyes (Marshall, 1982). As previously pointed out, either analog or digital filters are employed in signal processing. For example, antialiasing analog filters are employed before the signal is digitized (see the

Instrumentation

167

discussion of the APF process, Section VIII.C.3). Digital filters, as discussed here, involve computer methods which operate on a set of numerical data. Probably the simplest way to apply a filtering process to digitized Raman data is first to Fourier transform the data and then to perform the desired operation to reject spurious frequencies. Then, the inverse Fourier transform is performed to restore the original Raman spectrum with an appreciable improvement in the S/N. Due to the high speed of the FFT algorithm to evaluate the direct and inverse discrete Fourier transforms (DFT), such a filtering process is computationally efficient. However, factors such as the finite number of points, the noise level and the truncation of the Fourier spectrum require some consideration. Thus, some caution must be exercised when this filtering process is employed. The following example illustrates some of these difficulties and the way in which they can be remedied. Consider the low-pass digital filtering of the set of numbers Sn = s{ni^v), as shown in Fig. 59. In the Fourier domain the DFTs of the input 5(x), and output, Z(x), are related by Z{x) = H{x)'S{x),

(129)

where H{x), the transfer function, is a boxcar whose characteristics depend on the chosen frequency rejection.* Thus, with the use of the characteristics of the transfer function shown in Fig. 59, all of the frequency components above /3 cm are set equal to zero. An inverse DFT will then generate the filtered signal which, as it appears in Fig. 59, exhibits oscillations and distortions. Indeed, truncation of the Fourier spectrum with the use of a rectangular function corresponds in the v domain to a convolution with the appropriate sine functions (sin27rj8F)/7ri^), leading to oscillations or 'feet' on either side of a Raman band. The spurious features are sometimes referred to as 'Gibb's ears'. The truncation by a rectangular function results in errors in the Raman spectrum which affect the band intensities. Furthermore, the feet often interfere with the observation of nearby weak Raman bands. Fortunately, these problems can be overcome by apodization (from the Greek: aTTov<;, without feet) with the use of other truncation functions (windows) in the Fourier domain. Various so-called apodizing functions are *It should be noted that Eq. (129) corresponds in the 1^ domain to a convolution product which can be written in discrete form as

^n=2j^k

(130)

This equation is the general formula for nonrecursive digital filtering. In the present example of a low-pass digital filter with the transfer function shown in Fig. 59, the coefficients h^. are given by hi^ = (sinlTr^pJ/Trvf^), with vi^ = k!iv. In principle, an infinite number of filter coefficients is required to obtain the same result as in the process depicted above which used the direct and inverse FTs. In practice at least 100/z^^ weights are necessary and, therefore, this type of digital filtering is not considered to be very efficient.

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M. Melhaye et al.

-^(v)

h

DIRECT FT

A y PK

Vi

(cm)

(cm ')

^Av H{x)

Figure 59 Low-pass digital filtering with the use of the FFT showing the effects of (a) rectangular truncation and (b) Hanning apodization.

Instrumentation

169

available (triangular, Hamming, Manning, Blackman, Connes, etc., see Harris, 1978; Miquel, 1985) which can reduce the effect of truncation errors by making the transition in H{x) between one and zero less abrupt than those produced by the rectangular function. In this way the feet can be greatly reduced in intensity. However, this result is achieved at the expense of spectral resolution, as shown in Fig. 59 for the case of a Hanning window. In general, apodization always invokes some compromise with respect to both resolution and band shape.

3. Data Smoothing Smoothing has been known for a long time and modern spectroscopists are familiar with least-squares polynomial smoothing (LSP), better known as Savitzky-Golay smoothing (Savitsky and Golay, 1964). A great deal of literature has been devoted to LSP smoothing (Madden, 1978) and to the distortions brought about by this method of data treatment (Marchand and Marmet, 1983). Moreover, many spectrometer manufacturers provide Savitsky-Golay smoothing as part of the software package. Surprisingly little attention has been paid to a very efficient and relatively 'harmless' algorithm known as 'binomial smoothing' which was recently examined in detail by Marchand and Marmet (1983). This technique, which is presented below, has proved very powerful and has been used extensively in our laboratory. It has recently been implemented in new-generation Raman spectrometers (Aubard et al, 1989). The smoothing process generally refers to a special class of filtering, namely low-pass filtering. From Eq. (130) smoothing is assumed to be a linear weighting of the input data values, 5„ (AZ = 1, . . ., TV). The smoothed output data Z„, are given by +y Sn~kak. (131)

^«= k=-j2

where 2/ + 1 is the number of coefficients (points) in the filter (commonly known as the smoothing window) and k is any positive integer. The aj^ values are the filter coefficients which obey the normalization condition +]

k=~j

Thus, in LSP smoothing mentioned above, a least-squares fitting is made of a set of 2] + 1 data points to a polynomial of degree /i, where h<2j+l. For cubic or quadratic polynomial smoothing, for example, the Uj^ values are calculated from the relation (Madden, 1978) aj, = 3(3/2 + 3y _ 1 _ se)l[{2j + 3)(2/ + 2)(2/ - 1)].

(133)

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M. Melhaye et a/.

o



1500

1000

500

WAVENUMBER (cm~^)

Figure 60 Binomial smoothing of a Raman spectrum of the elUpticine-DNA complex adsorbed on silver colloids (514.5 nm, 100 mW). (a) Unsmoothed spectrum; (b) 11-point binomial smoothed spectrum (i.e. five times the three-point binomial smoothing). In binomial smoothing the coefficients, a/^, are the binomial coefficients (divided by their sum), which are easily obtained from Pascal's triangle. Moreover a (2p + l)-point binomial filter is easily computed by simply repeating p times the three-point binomial filter. Thus, from Eq. (131), [7 = 1; «-i,o,i = (1,2, l)/4] and the basic three-point binomial smoothing equation for each point n of the signal (except for the first and last points of the data set) is given by 4^n-l ~^ l^n + A^n + \'

(134)

This operation may be repeated p times to obtain the desired smoothing; e.g. to achieve 21-point smoothing the three-point smoothing operation must be repeated ten times. Binomial smoothing is easy to program in any language and is very rapidly executed by the computer if the data are composed of integers, since the divisions by two and four can be replaced by arithmetic right shifts. As demonstrated in Fig. 60, this method is particularly efficient for noisy Raman

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spectra which exhibit rapid variations and narrow bands. Moreover, it completely eliminates the phase rotations and overshoots which can occur with Savitsky-Golay smoothing when high-order filter windows are used. F. Conclusion

A brief, but hopefully substantial, survey of digital analysis and processing has been presented in this chapter. Thus, you now know a httle more about the most important mathematical tools and principles and can appreciate the utility and efficiency of any popular signal analysis and processing method. Never forget that digital data treatments when unsuitably apphed lead to side effects which alter experimental data. 'Lost information cannot be retrieved', as my mountain chmber colleague D. Taupin (1988) said in his noteworthy book Probabilitiesy Data Reduction and Error Analysis in the Physical Sciences. With that maxim in mind, it's now up to you . . .

REFERENCES Alarie, J. P., Stokes, D. L., Sutherland, W. S., Edwards, A. C. and Vo-Dinh, T. (1992). AppL Spectrosc. 46, 1608. Asher, S. A., Flaugh, P. L. and Washinger, G. (1986). Spectroscopy 1, 26. ASPEN (1970). G. Vanasse (ed.), Conf, Fourier Spectrosc. Airforce Cambridge Research Laboratory, Bedford, MA. Aubard, J. (1980). Doctoral thesis, Paris. Aubard, J., Levoir, P. and Delamar, M. (1989). In: DILOR XY Software Notice. Lille, France. Barbillat, J. and Chapput, A. (1990). Proc. SPIE 1341, 233. Barbillat, J., Da Silva, E. and Roussel, B. (1991). /. Raman Spectrosc. 22, 383. Barbillat, J., Da Silva, E. and Hallaert, J. L. (1993). /. Raman Spectrosc. 24, 53. Battey, D. E., Slater, J. B., Wludyka, R., Owen, H., Pallister, D. M. and Morris, M. D. (1993). Appl. Spectrosc. 47, 1913. Bergin, F. J. and Shurvell, H. F. (1989). Appl. Spectrosc. 43, 516. Bousquet, P. (1969). Spectroscopic instrumentale. Dunod, Paris. Bracewell, R. N. (1968). The Fourier Transform and its Applications, 2nd edn. McGraw-Hill, New York. Brenan, C. J. H. and Hunter, I. W. (1994). Appl. Optics 33, 7520. Brigham, E. O. (1974). The Fast Fourier Transform. Prentice Hall, Englewood Cliffs, NJ. Carrabba, M. M., Spencer, K. M., Rich, C. and Ranch, D. (1990). Appl. Spectrosc. 44, 1558. Chamberlain, J. (1979). The Principles of Interferometric Spectroscopy. John Wiley & Sons, Chichester. Chase, B. (1987). Anal. Chem. 59, 14. CNRS-ANVAR (1986). Patent no. 8604 947. CNRS-ANVAR (1987). Patent no. 8709 883.

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Pallister, D. M., Kei-Leeliu, K., Govil, A., Morris, M. D., Owen, H. and Harrison, T. R. (1992). Appl Spectrosc. 46, 1469. Plaza, P., Nguyen-Quy Dao, Jouan, M., Fourier, H. and Saisse, H. (1986). Appl. Optics IS, 3448. Portfield, D. and Campion, A. (1988). / . Am. Chem. Soc. 110, 408. Puppels, G. J., Collier, W., Olminkhof, J. H. F., de Mul, F. F. M., Otto, C. and Greve, J. (1991). / . Raman Spectrosc. 22, 217. Rosasco, G. J. (1980). In: R. J. H. Clark and R. E. Hester (eds), Advances in Infrared and Raman Spectroscopy. Hey den and Sons, London, vol. 7, pp. 223-282. Rosasco, G. J., Etz, E. S. and Cassatt, W. A. (1975). Appl. Spectrosc. 29, 396. Savitzky, A. and Golay, M. J. E. (1964). Anal. Chem. 36, 1627. Sawatzki, J. (1991). Fresenius J. Anal. Chem. 339, 267. Sawatzki, J., Lehner, C. and Kawai, N. T. (1994). Proc. XXII EUCMOS, Essen, Germany, p. 220. Schrader, B., Baranovic, G., Keller, S. and Sawatzki, J. (1994). Fresenius J. Anal. Chem. 349, 4. Schwab, S. C. and McCreery, R. L. (1984). Anal. Chem. 56, 2199. SELFOC (1987). Technical Document, Nippon Sheet Glass Co., Somerset, NJ. Sheppard, C. J. R. and Min Gu (1991). Appl. Optics 30, 3563. Singer, R., Knoll, P. and Kiefer, W. (1988). In: R. J. H. Clark and D. A. Long (eds), Proc. Int. Conf. Raman Spectrosc. (London). John Wiley & Sons, Chichester, p. 953. Solymar, L. and Cooke, D. J. (1981). Volume Holograms and Volume Gratings. Academic Press, London. Sommer, A. J. and Katon, J. E. (1991). Appl. Spectrosc. 45, 527. Sommer, A. J. and Katon, J. E. (1993). Spectrochim. Acta 49A, 611. Stammreich, H. and Forneris, R. (1961). Spectrochim. Acta 17, 775. Stearns, S. D. (1975). Digital Signal Analysis. Hayden, Rochelle Park, NJ. Strassberg, D. (1991). Electron DesigNers, July 18, 76-86. Taupin, D. (1988). Probabilities, Data Reduction and Error Analysis in the Physical Sciences. Monographies de Physique, les Editions de Physique, les UUs. Tilotta, D, C. and Fateley, W. G. (1988). Spectroscopy 3, 1. Tilotta, D. C , Freeman, R. D. and Fateley, W. G. (1987). Appl. Spectrosc. 47, 1280. Turner, P. H. (1994). Bruker Report 140, 36. Walrafen, G. E. (1978). In: E. D. Schmid, R. S. Krishnan, W. Kiefer and H. W. Schrotter (eds), Proc. Vlth Int. Conf. Raman Spectrosc, BangaloYQ, India. Heyden, London, p. 500. WilHams, A. B. and Taylor, F. J. (1985). Electronic Filter Design Handbook, 2nd edn. McGraw-Hill, New York. WilHams, K. P. J. (1990). / . Raman Spectrosc. 21, 143; 147. Wilson, T. and Sheppard, C. (1984). Theory and Practice of Scanning Optical Microscopy. Academic Press, London. Woods, T. N., Wrigley, R. T., Rottman, G. J. and Haring, R. E. (1994). Appl. Spectrosc. 33, 4273. Yang, B., Morris, M. D. and Owen, H. (1991). Appl. Optics 45, 1533. Zhong-Yuan Zhu and Yappert, M. CeciHa (1992a). Appl. Spectrosc. 46, 912. Zhong-Yuan Zhu and Yappert, M. CeciUa (1992b). Appl. Spectrosc. 46, 919.

Raman Imaging Jacques Barbillat

I. INTRODUCTION For many years Raman spectroscopists have collected spectral data, either from a large amount of sample material or from a specific region, by focusing the laser beam onto a small area - sometimes determined by the diffraction limit of the light excitation. This technique yields information about the local composition of the sample, but does not provide an overview of the spatial arrangement of the various molecular species within a heterogeneous specimen. However, since Raman scattering involves visible radiation, the spatial distribution of a given compound may be obtained by means of optical methods which are usually referred to as Raman imaging techniques. All of them are based on the same general principle, which consists of mapping the spatial arrangement of one constituent of an inhomogeneous sample by isolating from the Raman scattered photons those which originate from a characteristic Raman line of the specific compound (Fig. 1). In fact twodimensional and three-dimensional Raman imaging are not simple due to the inherent weakness of Raman scattering; therefore relatively sophisticated techniques are required. Although Raman imaging is not limited to the study of microsamples, most techniques currently available are in the domain of Raman microscopy. The present discussion will be limited to this field. Basically, Raman imaging methods can be classified in two categories, referred to as 'parallel- or direct-imaging' or 'series-imaging' techniques. (i) The direct-imaging technique results in the immediate production of a complete two-dimensional (2D) image at a chosen wavelength which is characteristic of a molecular compound within the fully illuminated specimen. It necessarily requires a 2D detector (SEC* or SITf cameras were used in the early days of Raman microscopy, but are now advantageously replaced *Secondary electron conduction, tSilicon intensifier target.

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Figure 1 Principle of Raman imaging. by CCD detectors). Direct imaging does not necessitate additional data processing to visualize an image, although image quality can be improved with the help of sophisticated data treatment. (ii) On the other hand, series-imaging techniques require image reconstruction, which is achieved either by scanning the sample with a finely focused laser beam or by encoding with a mask the image of the sample illuminated by an expanded laser beam. Although single-element detectors can be used for image reconstruction, the best results are obtained, again, with 2D multichannel detectors such as a CCD. II. DIRECT IMAGING A. Global Sample Illumination Before entering the microscope, the laser beam is expanded to fill the field of view of the microscope objective. This expansion can be obtained by

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slightly defocusing the laser spot in the image plane of the objective in order to cover a larger portion of the sample in the object plane. However, this arrangement results in a Gaussian intensity distribution at the sample, which reflects the profile of the laser beam, with a maximum hght intensity at the center and very httle at the outer edge. This drawback can be overcome by overfilhng the field of the microscope with the laser radiation. However, this approach reduces the laser irradiance at the sample. Whatever the situation, the sample is not correctly illuminated because of the speckle noise caused by the coherence of the laser beam. A moving ground glass diffuser can reduce the coherence of the beam but, again, with a significant loss of intensity. Wide-field illumination can also be achieved by projecting on the sample, with the use of the microscope objective, an enlarged image of the exit tip of an optical fiber hnked to the laser source (ElHs, 1979). The fiber must be vibrated in order to ehminate the laser speckles and to obtain uniform illumination of the sample. Aperture-scanning illumination also provides wide-field illumination. It consists of scanning the condenser or objective aperture with a focused laser beam (Ellis, 1988). Then, at any time, the field of the microscope is filled by a laser beam originating at the aperture point and the sample is illuminated without speckles at successively changing tilt and azimuth angles. Alternative methods can consist of axicon-type optics or a rotating device to feed the annular condenser of a dark-field microscope objective with the laser beam (Delhaye and Dhamelincourt, 1975). The latter method is preferable since it also provides laser-speckle elimination.

B. Spectral Filtering

The scattered radiation from the different regions of the specimen is collected by the microscope objective and one Raman line which is characteristic of the constituent to be imaged must be filtered out by a stigmatic wavelengthselection device before being received by a 2D detector. Various instrumental configurations have been proposed that differ in the way in which the desired wavelength is selected. The diffraction-grating spectrometer is a well-proven tool and, as such, was retained in the first design (MOLE microprobe) introduced 20 years ago by Delhaye and Dhamelincourt (1975). This Raman microscope comprised a double-additive monochromator with concave diffraction gratings which acted as a bandpass filter (Fig. 2). By properly couphng the image plane of the microscope objective to the surface of the grating and the exit pupil of the microscope objective to the entrance slit of the spectrometer, goodquality images which preserve the spatial resolution of the microscope are transmitted through the spectrometer to the detector (intensified SEC or SIT camera). However, this optical conjugation has a direct effect on the spectral

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resolution of the spectrometer, since the slit width must be at least equal to the size of the image of the objective pupil projected onto the slit. A reduction of the slit width below this limit leads to apodization of the spread function of the microscope. As a result, the spatial resolution of the final image will be lowered. The spectral resolution that could be achieved with the MOLE microscope, without loss of spatial resolution, was typically 16cm~^ for a x20 objective and about 12cm~^ for a x40 objective. Less complicated filter-based monochromator systems are now available. Batchelder et al. (1991) have developed an imaging instrument in which the spectral filtering is accomplished with the use of a set of interference bandpass filters which yields a spectral resolution of about 20cm~^ (Fig. 3). Efficient rejection of the laser light is obtained with a Raman notch holographic filter which exhibits high transmission outside of the rejection band. A dedicated computer ensures the selection and proper angle tuning of the dielectric filter. It is adapted to the desired wavelength and also handles the CCD controland data-acquisition parameters. Typical image dimensions are in the range 10-200 |xm, with a spatial resolution of about 1 ixm. Puppels et al. (1993) have associated a fixed position, narrow-band, dielectric transmission filter and a tunable dye laser (Fig. 4). The selection of a given Raman band is obtained by tuning the dye laser until the wavelength of this Raman band coincides with the transmission peak of the filter. This configuration provides a spectral resolution of about 10 cm~^ around 690 nm. It results in a high-throughput, compact instrument with no

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moving parts. A set of Raman holographic edge filters helps to eUminate most of the laser light in the detection compartment. The key element in the Raman microscope developed by Treado et al. (1992) is a recently introduced acousto-optic tunable filter, which can replace a set of filters or a grating (Fig. 5). With this entirely solid-state installation, with no moving parts, Raman images can be collected with a CCD detector at a moderate spectral resolution of about 30 cm~^. Here again, a Raman notch holographic filter serves to reject most radiation at the laser frequency that could perturb the detection of weak Raman features. A new instrumental development which integrates a volume-phase, holographic transmission grating whose grooves are generated throughout the volume of a thick recording film is described by Battey et al. (1993) and illustrated in Fig. 6. A more complete description of volume holographic optics is given by Tedesco et al. (1993). An instrument might be conceived which integrates a tunable Fabry-Perot etalon and a fixed or a tunable bandpass filter which passes only one order of the etalon (Meriaux et al., 1971). By tuning both filters it should be possible to select any Raman line in the spectrum and obtain high-resolution images.

C. Characteristics of Direct-imaging Techniques 1. Advantages The laser power at the sample is distributed over all of the pixels of the sample. If each position of the sample is illuminated with the maximum allowable power, a very high-power laser is required. The advantage is that the signal collection time is reduced with respect to point illumination for a comparable S/N. This result is called the distribution advantage of the direct-imaging technique, which provides relatively short acquisition times.

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2. Disadvantages Only one piece of spectral information can be obtained at a given time. Access to other wavelengths requires additional acquisitions at different settings of the spectral filtering device. Generally, one single frequency image is not sufficient to establish unambiguously the presence of a given molecular species because the signal detected at that frequency could also correspond to a fluorescence background. Three measurements are thus necessary to confirm the presence or not of a constituent: one at the expected Raman shifted frequency and one on each side of this position. In the case of a strong fluorescence background that masks the Raman signal, all three images will appear to be quite similar. In the case of filter-based instruments, the use of notch filters to suppress the laser radiation Hmits the access to the low-wavenumber region. As the filter characteristics are defined at the conception of the instrument, the spectral resolution cannot be adjusted to optimize the detection conditions for each application.

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In principle, direct-recording imaging techniques cannot benefit from the confocal arrangement. However, significant improvement of the axial resolution can be achieved through sophisticated data processing such as the nearest-neighbor deblurring technique (Govil et al., 1991) or constrained iterative image restoration (Govil et al., 1993).

III. SERIES IMAGING Laser-scanning and the global illumination Hadamard encoding method belong to this family of series Raman imaging techniques, which is based on image reconstruction. If all spatial and spectral information is to be recovered, a set of 2D (or 3D) images of the sample at various wavelengths can be visualized and manipulated only after the collected data have been processed.

A. Laser-scanning Methods 1. Point Illumination The point-illumination Raman technique is based on the same principle as the well-known laser fluorescence confocal microscope. A laser spot is scanned over the sample sequentially in a raster pattern (or the sample is moved equivalently under the microscope objective). At each resolved position of the sample a signal is collected by the microscope objective, spatially and spectrally filtered, then focused on a single-element detector (photomultiplier tube). However, as the Raman intensity is typically several orders of magnitude less than the fluorescence signal of most specimens studied by fluorescence methods, the collection of a complete set of spatial/spectral Raman data is very time-consuming. This technique should be reserved for the imaging of a very small region of the sample. Multichannel detection can help to increase the amount of data collected at the same time. For a given sample position, a linear-array detector can record, at the exit port of a dispersive spectrograph, a large number of spectral elements, thus reducing the time needed to collect a multiwavelength spatial representation of the sample (Delhaye et al., 1982). 2. Line

Illumination

A further reduction in data collection time is achieved with the use of 2D detectors and sample illumination along a narrow fine which is optically conjugate to the entrance sHt of a stigmatic spectral analyzer (Fig. 7). With

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this configuration the entire active area of the 2D detector can be exploited. One dimension of the CCD, perpendicular to the slit, is used to collect (from each point of the line) spectral information, while the other dimension, parallel to the slit direction, yields spatial information (intensity profiles). Sample illumination is achieved by laser deflection: a scan (galvanometer) mirror vibrating at a few cycles per second deflects the laser beam along a narrow line in the image plane of the microscope. As the light spot is moved in the image plane, the conjugate light spot, which moves in the object plane, scans the sample. In order to illuminate the objective correctly, an intermediate optical system (field lens) forms an image of the pupil of the microscope objective on the scan mirror. In an alternative method the laser beam is spread in one direction with a cyhndrical lens that illuminates simultaneously all of the points in a rectangular region of the sample. However the intensity distribution along the line is no longer uniform, as it reflects the Gaussian profile of the laser beam.

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The Raman light scattered by the sample is collected with the microscope objective and passed through the beamsplitter that is used for the separation of the excitation and detection paths. It is then focused in a Hne onto the entrance slit of the (astigmatic-free) spectral analyzer. Within a single exposure, the CCD detector in the exit focal plane collects a series of spectra from all of the spatially resolved regions of the sample along the laser line. In other words, the CCD detector collects a series of intensity profiles which represents the spatial distribution of the various constituents of the sample along the laser line. Sequential translation of the microscope stage perpendicular to the line-scanning direction allows computer recording of a complete set of spectral data for every point of the specimen. The collected spatial/spectral information can be converted into selective images of the distribution of the chemical species present in the sample. Barbillat (1983) has obtained Raman intensity profiles with a modified multichannel Raman microprobe with the use of a galvanometer optical scanner to illuminate the sample and a linear photodiode-array detector positioned perpendicular to the spectral dispersion. Bowden et al. (1990) have designed a line-scanned micro-Raman spectrometer which uses a CCD imaging detector to obtain intensity profiles from a 100-|xm-long line (Fig. 8). Laser scanning is achieved by a moving lens. The line-scanning arrangement provides better depth resolution and improved optical sectioning than does the direct-imaging device. The narrow

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entrance slit acts as a rectangular diaphragm which reduces the intensity of the light originating from regions of the sample that are not in focus. However, optimum rejection would involve the use of a pinhole diaphragm, an element which is not compatible with the line-scanning optical layout.

3. Confocal Line Scanning The limitation indicated in the previous paragraph can be overcome with the use of the Confocal Raman Lme Scanning (CORALIS) imaging system proposed by Barbillat et al. (1992). The CORALIS system, which is based on a dual-scanning device (Delhaye et al., 1992), benefits fully from the confocal arrangement, while providing Hne illumination of the sample and 2D-CCD detection. The basic confocal line-scanning system is illustrated in Fig. 9. The laser beam is focused on a first pinhole diaphragm, where spatial filtering takes place. The 'cleaned-up' laser beam then passes to a galvanometer-mirror, driven with a triangular waveform, that scans the laser spot in the image plane of the microscope objective. The objective projects a narrow laser line on the sample. For each tilt angle of the scanning mirror there corresponds a specific point at the sample from which a Raman-scattered beam is collected by the objective and returned to the spectrograph through the same optical elements as the laser beam. The scattered radiation and the incident laser beam follow the same path in opposite directions and are separated only after the scattered beam has passed the scanning mirror. The essential feature of this system is that after passing the scanning mirror the scattered beam is at rest (as is the laser beam), independent of the tilt angle of the scanning mirror, i.e. whatever the position of the laser beam on the sample. After passing through the beamsplitter, which separates the incident and detection paths, the scattered beam is brought to a focus. As pointed out above, this focal point is unique whatever the position of the laser beam on the sample. In fact, this focal point is optically conjugate with every point of the sample along the illuminated area, as well as with the first pinhole on the laser beam. The result is that the Raman signal can be filtered with a pinhole diaphragm located at the focal point which acts like the confocal pinhole of the confocal scanning microscope (Minski, 1961). The size of the diaphragm aperture can be adjusted to monitor the axial resolution, depending on the geometry of the sample. Behind the confocal pinhole the Raman signal is deflected again, this time along the slit height of the spectrograph, by a second scanning mirror absolutely synchronous with the first one. Its scan amplitude, which can differ from that of the first one, is adjusted so that the Raman signal emerging from the confocal pinhole entirely covers the columns of the CCD detector in the focal plane of the stigmatic spectrograph. As a result, each row of the CCD

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Figure 9 Optical diagram of the confocal, line-scanning imaging system. detector is conjugate with one point of the sample, and each pixel of a row is an image of the conjugate point at a given wavelength. Within a single exposure the CCD collects a complete set of Raman intensity profiles depicting the spatial distribution of the different chemical species present along the scan line. This configuration is not unique and, for instance, the same result can be achieved with the second scanner located at the exit of

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the spectrograph, so as to scan directly the CCD detector. Whatever the configuration adopted, complete spatial exploration of the sample is performed by moving the microscope stage stepwise in a direction perpendicular to the scan line. At each step a set of multiwavelength intensity profiles is stored in a computer. Single-wavelength 2D images are generated by processing the huge collection of spatial/spectral data accumulated by the computer. This configuration exhibits original features that arise from the fact that the scan mirrors can be driven separately, namely, (i) The entire area of the detector is always optimally exploited whatever the length of the scanned region of the sample, as Raman intensity profiles of this region are always projected over the full height (columns) of the CCD. In other words the spread factor, defined as the number of pixels of the detector per unit length (micron) at the sample, varies each time the laser scan angle is modified. The direct consequence is that the spatial definition of the Raman image, which is directly related to the spread factor, can be improved by simply reducing the length of the laser scan fine. However, it has to be kept in mind that this improvement does not concern the spatial resolution, which is always defined by the confocal arrangement. (ii) Depth profiles can be obtained by moving the sample stage back and forth along the optical axis of the microscope objective, while leaving the laser scan mirror at rest. (iii) Intensity profiles can be recorded vs. the spatial position of the laser spot within the sample. Alternatively, intensity profiles can be obtained for a given position of the spot in the field of view of the microscope, as a function of any physical parameter (i.e. temperature, pressure, etc.) by leaving the laser beam at rest on the sample and scanning the Raman signal on the slit. A commercial instrument based on the CORALIS principle has been developed by DILOR (1992). 4. Common Characteristics of Scanning Imaging Techniques Line illumination techniques generally involve the use of a grating monochromator to disperse the Raman spectrum on the CCD detector. Basic configurations comprise either a single-stage spectrograph with a Raman notch filter to cut off the Rayleigh fine or a triple monochromator. The latter configuration allows access to the low-frequency spectral region. Line illumination may result in better spectral resolution (without signal reduction) and stray light rejection than direct imaging, as a narrow line of scattered fight is projected on the entrance slit of the dispersive spectrograph. Typically, this configuration results in slit widths as small as a quarter or a third of the width of the laser fine in the image plane of the microscope,

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i.e. 25-33 juim, which corresponds to a spectral resolution of about 3 cm ^ with a 500 mm single-stage spectrograph.

B. Encoding Techniques Multiplexing is a well-established method which is used to improve the collection of spectral data, including images. Instead of being dispersed on a multielement detector, the spectral or spatial elements are encoded with a mask before reaching a single detector; the original data are recovered by mathematical treatment of the complete set of data obtained at different positions of the mask. Although Fourier and Hadamard transforms are two closely related linear transformations which are used in most spectroscopic multiplexing, only the Hadamard technique has been employed to obtain Raman images. This method permits global illumination of the sample, as in direct imaging techniques, and can be used to obtain multidimensional imaging with a single-element detector. Treado and Morris have developed several Raman spectroscopic systems that exploit the spatial multiplex advantage of Hadamard imaging. They are capable of delivering 2D, spectral images at a spatial resolution close to the diffraction limit. In the initial system (Treado and Morris, 1989) 2D Raman images were multiplexed into a single-channel detector with a two-dimensional 255-element Hadamard mask. Spectral selection was achieved by a monochromator that isolated a single Raman line. In a subsequent design (Treado and Morris, 1990) one-dimensional spatial encoding was combined with a Hadamard mask and multichannel spatial and spectral detection with the use of a CCD detector (Fig. 10). In the image plane of the microscope the magnified image of the sample was spatially encoded in only one direction {x for instance) with a one-dimensional mask consisting of slots elongated along the y direction. A cylindrical optical system with its generatrix parallel to the slots of the Hadamard mask compressed the encoded x direction into one pixel and left the y spatial information unchanged. The x-y image was converted into a narrow ^'-figure conjugate to the entrance sUt of a dispersive stigmatic spectrograph. For each step of the encodement sequence (which may consist of up to 256 settings of the mask) the CCD detector collects in the focal plane of the spectrograph >^-intensity profiles versus spectral information. The x spatial information that has been encoded by the Hadamard mask is retrieved by carrying out the inverse, fast-Hadamard transformation (FHT) of the data for each pixel. After computation, a complete set oi x,y,n data stored in the computer can be used to generate single-wavelength images. Usually, only a few wavelengths are needed to describe the specimen. Thus, images are generated more rapidly, as the inverse FHT is performed with only a Hmited number of pixels.

Raman Imaging 189 Cylinder Lens

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Figure 10 Multichannel Hadamard-transform Raman microscope. In the case of the Hadamard multichannel system, the sample as well as the laser beam are fixed during the measurement; only the mask is moved, in order to achieve spatial encoding of one spatial direction. As the displacement takes place in the image plane of the microscope, there is no need for fine, accurate and reproducible micrometric displacement, as required by scanning systems. The Hadamard technique provides the multiplex advantage (Fellgett) when the instrument is detector-Umited, i.e. when the main source of noise is the detector. The installation with Hadamard encoding and CCD detection behaves differently, as the CCD detector produces very low noise. Thus, the 'photon' (or shot) noise due to the total Hght flux may become preponderant. Then, the advantage of this technique is reduced compared to others such as the scanning methods discussed below.

IV. SIGNAL-TO-NOISE RATIO (S/N) AND COLLECTION TIME In this analysis a very simple model will be assumed in which the detector readout noise is negligible compared to the signal noise. This condition is

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correct in the case of the CCD detectors considered here. It is further assumed that jc-point by >'-point images are studied and the smallest spatial element on the sample is conjugate to a single pixel of the CCD detector. The maximum allowable laser power at the sample is n photons second per spatial element. The total duration for collecting M images at M different wavelengths is T seconds for every method. The background noise is neglected. A. Line Illumination versus Global Illumination 1. The Same Total Laser Power Used in Both Measurements (a) Line illumination The signal originating from a point arises from kn TIxy photons, where k depends on the nature of the sample. This result yields the relation S^ = kK7]nT/xy,

(1)

where S^ is the number of charges at the CCD output mode. The overall optical efficiency determines the value of K and the quantum efficiency of the CCD detector is given by h. The associated noise can be expressed by N,= {kKr)nTlxy)^'^.

(2)

Thus, the signal-to-noise ratio is equal to S,IN, = (kKrjn TIxy) ^'^.

(3)

Actually, the time available for each point is less than TIxy, as the fine has to be moved y times onto the sample in order to scan all of the area to be imaged. (b) Global illumination In this case the magnitude of the signal originating from a point is given by k{nlxy){TIM), which leads to the expression S^ = kK7]nTlxyM.

(4)

The associated noise is equal to yVg = {kKr^nTlxyMY'^.

(5)

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for the signal-to-noise ratio. Thus, global illumination never leads to a better value of S/N, but the sample is in this case well protected against laser damage, as the local irradiance is very low. 2. Each Pixel of the Sample Receives the Maximum Allowable Power in both Measurements (a) Line

illumination

The situation is unchanged, since each point always receives the maximum allowable power, as in the first calculation. The signal-to-noise ratio is still given by {kKiqnT/xyy^^. (b) Global illumination Now, the magnitude of the signal originating from a point is equal to nT/M, leading to the relation 5g = kKr]nT/M.

(7)

The associated noise level is then given by A^g = {kK7]nTIMf'^

(8)

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S,IN^.

(9)

In the real situation the background is seldom negligible, so that correct signal evaluation often requires background subtraction. This operation reduces the time available for each point, as a second image (at least) has to be acquired at a neighbouring wavelength. As a consequence, the S/N is divided by a factor of two, as pointed out by Puppels et al. (1993). This result means that the S/N in global imaging is better than that in the line-scanning technique, as long as the number of desired images is kept smaller than half of the total number of pixels in the image.

V. HADAMARD IMAGING VERSUS GLOBAL ILLUMINATION AND LINE SCANNING A comparison of these techniques is given by Puppels et al. (1993). It is found that in general direct imaging gives a better S/N and that Hadamard imaging

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is to be preferred to direct imaging only in the case of very low signal levels. Moreover, Hadamard imaging does not offer a performance that is superior to Hne-scanning image reconstruction. The reason is that the general Fellgett advantage of the Hadamard multiplexing technique is not achieved when the detector exhibits very low noise, as with the use of a slow-scan CCD detector operating at very low temperatures. The global-illumination techniques provide much more total laser power at the sample than do scanning techniques, although each pixel of the sample does not receive more energy. This result is called the distribution advantage. However, is it a real advantage in the general case? It is not evident that the sample can sustain so much power without damage, as the thermal relaxation is different when a sample is excited point by point or as a whole. The conclusion is that the S/N improvement discussed above is, in practice, certainly lower than the calculated value. Another way to benefit from the distribution advantage of global imaging is to reduce the time required to obtain a set of single-wavelength images, without improving the S/N. The rapid capture of survey images might be ultimately one of the best features of the direct-imaging techniques.

VI. EXAMPLES OF APPLICATIONS Figures 11-17 present some images obtained with the different systems described above. Figure 11 shows the Raman image of a sulfur inclusion within a host matrix of natural strontium sulfate (celestine) obtained with the MOLE microscope (Dhamelincourt and Bisson, 1977). Raman intensity profiles at various wavenumbers on a zircon material (Bowden et al., 1990) are displayed in Fig. 12, while Fig. 13 presents bright-field and Raman images at 998cm~^ of polystyrene spheres (Puppels et al., 1993). Figure 14 illustrates the direct recording of the Raman image of a thin film of rubber-toughened epoxy resin at 1665cm~^ (Garton et al., 1993). The confocal line-scanning image of a polymer film containing polypropylene and polyethylene is shown in Fig. 15. These two polymers are impossible to localize by visual inspection. CORALIS images are obtained with the dedicated software 'Spectrimage' developed by Sharonov and Manfait (1992). The Hadamard imaging of edge-plane microstructures in highly ordered pyrolitic graphite electrodes is illustrated in Fig. 16. The Raman image is reconstructed from the Raman band at 1360cm~^ (Treado et al., 1990). Figure 17 presents images obtained with an acousto-optical imaging system (Treado et al., 1992). They are images of a mixture of dipalmitoylphosphatidylchohne (DPPC) and L-asparagine aggregates which serves as a model for the study of lipid/peptide interaction.

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Figure 11 Raman image of a sulfur inclusion within a host matrix of natural strontium sulfate (celestine). (a) White-light image; (b) 473 cm~^ Raman image of sulfur; (c) 1000 cm~^ Raman image of SrS04.

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Figure 13 (a) White-light image of polystyrene spheres labeled with a fluorescent dye. (b) 998 cm"^ Raman image of polystyrene, (c) 515 nm fluorescence image obtained with blue excitation (420-430 nm).

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Figure 14 Raman image of a thin film of a rubber-toughened epoxy resin at 1665 cm-1.

VII. CONCLUSION In this brief chapter several techniques of Raman imaging have been reviewed and their performances compared. It is clear that there is no absolute answer to the question: What is the best method of imaging, direct scanning or encoding? None of the techniques described above can solve by itself all of the problems encountered in the field of Raman imaging. Depending on the desired result one technique may be preferred to another. It must be decided which advantage is desired in a particular application. If rapidity is of primary importance, direct imaging is to be preferred over image reconstruction, because it provides the desired information in less time. On the other hand, improved spectral resolution, background rejection or optical sectioning capabilities are best achieved with the use of confocal techniques.

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Figure 16 Hadamard imaging of edge-plane microstructures in highly ordered pyrolitic graphite electrodes. (A) Reflectance image; (B) 1360 cm~^ Raman image.

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Figure 17 Raman images of a mixture of dipalmitoylphosphatidylcholine (DPPC) and L-asparagine aggregates.

REFERENCES Barbillat, J. (1983). Doctoral thesis, Lille. Barbillat, J., Delhaye, M. and Dhamelincourt, P. (1992). Microbeam Analysis, 1514. Batchelder, D. N., Cheng, C , Miiller, W. and Smith, B. J. E. (1991). Makromolekulare Chemie-Makromolecular Symposia 46, 171-179. Battey, D. E., Slater, J. B., Wludyka, R., Owen, H., Pallister, D. M. and Morris, M. D. (1993). Appl Spectrosc. 47, 1913. Bowden, M., Gardiner, D. J., Rice, G. and Gerrard, D. L. (1990). /. Raman Spectrosc. 21, 37. Delhaye, M. and Dhamelincourt, P. (1975). /. Raman Spectrosc. 3, 33. Delhaye, M., Bridoux, M., Dhamelincourt, P., Barbillat, J., Da Silva, E. and Roussel, B. (1982). Microbeam Analysis, 275.

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Delhaye, M., Da Silva, E. and Barbillat, J. (1992). European patent No. 92400141.5. Dhamelincourt, P. and Bisson, P. (1977). Microscopica Acta 79, 267. DILOR (1992). Confocal Laser Raman Data Sheet. Lille. Ellis, G. W. (1979). / . Cell Biol. 83, 303a. Ellis, G. W. (1988). Proc. 46th Ann. Meeting EMSA. San Francisco Press, San Francisco, p. 48. Garton, A., Batchelder, D. N. and Cheng, C. (1993). Appl. Spectrosc. 47, 922. Govil, A., Pallister, D. M., Chen, L. H. and Morris, M. D. (1991). Appl. Spectrosc. 45, 1604. Govil, A., Pallister, D. M. and Morris, M. D. (1993). Appl. Spectrosc. 47, 75. Meriaux, J. P., Guttierrez, J. M., Schneider, C , Goutte, R. and Guillaud, C. (1971). Nouvelle Revue d'Optique Appliquee 2, 81. Minski, M. (1961). US Patent no. 3013 467. Puppels, G. J., Grond, M. and Greve, J. (1993). Appl. Spectrosc. 47, 1256. Sharonov, S. and Manfait, M. (1992). Personal communication. Tedesco, J. M., Owen, H., Pallister, D. M. and Morris, M. D. (1993). Anal. Chem. 65, 441. Treado, P. J. and Morris, M. D. (1989). Appl. Spectrosc. 43, 190. Treado, P. J. and Morris, M. D, (1990). Appl. Spectrosc. 44, 1. Treado, P. J., Govil, A., Morris, M. D., Sternitzke, K. D. and McCreery, R. L. (1990). Appl. Spectrosc. 44, 1270. Treado, P. J., Levin, I. W. and Lewis, E. N. (1992). Appl. Spectrosc. 46, 1211.

Raman Microscopy and Other Local Analysis Techniques Michel Truchet, Jean-Claude Merlin and George Turrell

I. INTRODUCTION

In Raman microspectrometry, as in any other method of local analysis, two main questions must be considered, namely: (i) How to localize the area to be analyzed, and (ii) How to analyze the area in question. To localize an area as small as 0.5-0.25 mm in size, the human eye is sufficient, but when the area is smaller than 0.25 mm, an optical system which is capable of delivering a magnified and contrasted image to the eye is necessary. Down to dimensions of ~0.5 juim, a photonic optical system can be employed, with the use of glass lenses and/or reflecting surfaces. Below 0.5 |ULm, the resolving power of photon optics is limited by the diffraction; thus, so-called corpuscular optics are required. They are usually electron optical systems. To analyze the area of interest the analytical system may be the same as that employed in the imaging technique, or quite different. In general, the two main characteristics of imaging and analyzing are related. These characteristics are: (i) The lateral resolving power, or lateral image resolution. Taking into account the lower limit for the human eye, i.e. approximately 0.25 mm, this condition defines the necessary magnification and the smallest area to be localized at the sample surface. (ii) The axial resolution or depth of field. Together with the lateral resolution, it determines the analyzed volume. It depends not only on the optical characteristics of the system, but also on the nature of the sample, i.e. the transparence for photons or the depth of penetration for electrons or ions.

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As a number of aspects of photon optics were presented in the previous chapter, this subject will not be considered here. However, some of the basic features of electron optics will now be briefly summarized (see, for example, Magnan, 1961).

II. ELECTRON MICROSCOPY A. Illuminating Column 1. Upper Portion of the Electron Microscope (a) The triode gun The electron beam is usually generated by a triode gun (Fig. 1). This is composed of a tungsten pin, electrically heated to white temperature - the cathode - which produces electrons by thermionic emission (cf. F o f Fig. 1). The anode is in the form of a ring maintained at a potential of several thousand volts with respect to the cathode {A of Fig. 1). The electrons, which are accelerated by this voltage, pass through the hole in the anode with the corresponding energy, generally measured in kiloelectronvolts (keV). Another annular electrode is placed between the cathode and the anode, the Wehnelt (W of Fig. 1), an adjustable voltage just below that of the anode, which forms an electronic lens that focuses the electrons at a point beyond the anode. As in photon optics, this 'point' is, in effect, a region in space where in this case the electron density is maximum. It is often called the cross-over. The cross-over is generally limited by a hole at the center of a diaphragm, which is considered to be the source of the optical system (Deo of Fig. 1). An LaB6 needle is often used as a cathode in place of the tungsten pin, as it has greater brightness. To enhance further the source intensity, a field-emission electrode is sometimes employed. It requires a very good vacuum, which is obtained with an ion pump. This source is more expensive and less rehable than the tungsten or LaB6 cathode; its use is therefore not as widespread. (b) Condenser stage Two or three lenses (Ci, C2, C3 of Fig. 1), which form at the surface of the sample (5 of Fig. 1) a reduced image of the cross-over, constitute the condenser stage of the column. These lenses are now electromagnetic, as electrostatic lenses, which are simpler but less reliable, have not been employed for some time. Similarly, permanent magnets are no longer used in magnetic lenses. The focal length of the electromagnetic lenses is adjusted

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Figure 1 Schematic diagram of the upper part of an electron microscope ('illumination'). F, filament; W, Wehnelt; A, anode; D^^, cross-over diaphragm; Q , C2, C3, electromagnetic lenses; S, sample.

by varying the voltages of the power supplies, and thus controlling the magnification of the entire stage. It should be pointed out that electromagnetic lenses are always convergent. In fact, the circular magnetic field is not the equivalent of a spherical lens, as there is only a radial component which creates the focusing. Thus, divergent magnetic lenses do not exist. It is perhaps evident that focusing with a three-stage condenser is better than with one which employs only two lenses. In the more recent electron microscopes, the two first lenses are conjugated in such a manner that the aberrations are reduced. There are two principal modes of application of the illuminating section. In the first case the convergence is Hmited and a wide area of the sample (of the order of several juim) is illuminated as homogeneously as possible. This mode is employed in the transmission electron microscope (TEM), which is described below. In a second case, the beam is as convergent as possible; the image of the cross-over is then an electron spot, referred to as the electron probe. In

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STE Figure 2 The diffusion volume and the electron-matter interaction. EB, electron beam; BE, backscattered electrons; RX, X-rays (characteristic and noncharacteristic - Bremstrahlung); SE, Secondary electrons; UVP, UV-visible photons; STE, straight-transmitted electrons (elastic interaction); DTE, diffused-transmitted electrons (inelastically scattered); S, sample; AE, absorbed electrons; DV, diffusion volume. scanning electron microscopy (SEM), a low-intensity electron spot is displaced over the surface of the sample to yield an image. In the analytical microprobe (see EPMA, below), its intensity is greater and the electron probe, generally static, permits elemental analyses to be made (see EDS and WDS). 2. Radiation-Matter

Interaction at the Sample (Eberhart, 1976)

Independent of the diameter of the electron beam {EB of Fig. 2), the impinging electrons, accelerated under several thousands of volts, interact with the atoms which constitute the sample (5 of Fig. 2). (a) Role of the electrons When the electrons are in the vicinity of the atoms, the positive charges of the nuclei modify their trajectories, resulting in an exchange of energy. After

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successive interactions, the trajectories become randomized. Finally, when the electrons have lost all of their energy, they are effectively absorbed by the sample. They are, then, the origin of the sample current. At each energy exchange, a photon is emitted {RX of Fig. 2); the energy of the photon depends on the quantum of energy exchanged. This particular emission is known as the Bremstrahlung. Its intensity depends upon the nature of the sample (i.e. the atomic numbers) and the energy of the incident electrons. After a few collisions, impinging electrons may emerge at the surface of the sample; they are the backscattered electrons {BE of Fig. 2). Their loss in energy is low and their penetration into the sample is generally limited. Their abundance is determined by the atomic numbers of the atoms in the sample. If the beam is normal to the sample surface, the backscattered electrons are distributed in a cone-shaped volume whose axis is coUinear with the optical axis, and thus perpendicular to the surface of the sample at the probe point. During electron-beam interaction, sample electrons can be ejected from the surface. They are the secondary electrons {SE of Fig. 2). Their energies are small, ~0-100 eV, and they originate from the more superficial layers of the sample. Their directions are randomly distributed throughout space. Among them are Auger electrons, which originate from atoms near the surface of the sample that are ionized by the incident electrons. The spectrum of the Auger electrons is determined by the atomic orbitals involved in the atomic transitions. It is thus characteristic of the atoms and can yield analytical information. Visible or UV photons are emitted from some samples. As they are characteristic of the sample, they can be used for molecular analysis. However, this cathode luminescence is still not widely employed as an analytical method. When the sample is thin, many electrons are transmitted. Some of them have not been scattered. However, others have undergone an interaction with the sample. The energy loss of the latter is characteristic of a given electronic transition. The analysis of this effect forms the basis of electron energy-loss spectrometry (EELS). It is, in principle, both elemental and molecular. Moreover, the role of the transmitted electrons which have been scattered is important, as the contrast they introduce from one point in the sample to another yields an image. This process is known as transmission imaging in EELS and the image produced is of analytical importance. (b) The electron microprobe During the interaction of an impinging electron with an atom, an atomic electron of a deep level (K, L or M) may be ejected. The atomic electrons

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of upper levels fall down in a series of de-excitation processes. All of the photons that are emitted at each transition have the same energy, which is determined by the atomic number of the atom. This kind of interaction gives rise to a general analytical method, the electron microprobe. 3. The Image In electron imaging, as in any other kind of imaging process, there are two principal methods. In the first, all of the electrons involved in the image formation are simultaneously collected. This mode of operation is mainly employed to obtain transmission images. In the second method each elemental part of the image, or pixel, is acquired one after the other with the use of a scanning system. The electron beam is finely focused in a small spot whose diameter (i.e. the reduced image of the cross-over) determines the size of the pixel. The electrons used to obtain the image may be due to transmitted, backscattered or secondary electrons. (a) The conventional transmission electron microscope

(TEM)

In this system the convergence, which determines the dimension of the cross-over image, is small and the illumination is conjugated with the image at the sample. The electromagnetic, optical imaging system beneath the sample consists of an objective, an intermediate lens and a projector (Fig. 3). It produces a magnified image of the illuminated part of the sample (or a smaller part of it, if there is a limiting-field diaphragm). This image is observed directly on a ZnS screen, recorded on a photographic plate or, more recently, acquired by a TV or CCD camera. To obtain a good image with the transmitted electrons, the sample must of course be very thin - the thinner the sample, the better the image. For example in biology, a chemically stable tissue included in a resin, or frozen, is sectioned at 50-80 nm. The resulting image is given by the contrast (intensity difference) from one point and another, depending on the magnitude of the scattering. If a point contains numerous heavy metals, such as Ag, Os, Pb, Au or U, the scattering is strong, whereas at points containing only the light elements of living matter (H, C, N, O, P, S) the diffraction is poor, i.e. electrons are numerous. For a sample devoid of heavy atoms, the contrast is given only by the density differences and is generally of inferior quality. The lower the accelerating voltage, the better the contrast; but for high magnifications, i.e. high resolution, the electrons are strongly accelerated. Thus, there is a trade-off between contrast and resolution which determines the choice of the accelerating voltage. It should be pointed out that, unUke glass lenses, magnetic lenses cannot

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be corrected for spherical aberration. Thus, the angular aperture (and hence the N.A.) is very small, typically, 10~^rad. The depth of field, or axial resolution, is very large, although this property is not important in the case of thin samples. The lateral resolution is always excellent because, even at low voltage, the associated wavelength is much shorter than that of a photon. For good instruments, a resolution of 1-2 nm is routinely obtained, which corresponds to magnifications as great as 100 000. Recent electron microscopes which employ very high voltages (200 kV-3 MV) yield very high resolution, but their main advantage is to permit the study of 'thick' specimens. For example, in biology, tissue sections up to 1 |xm can be investigated. However, 3D

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TED

Figure 4 Schematic diagram of a scanning electron microscope (SEM). EB, electron beam (finely focused on the sample surface); 5, sample; C, condenser; D, deviator (electrostatic plates or magnetic coils); BED, backscattered-electron detector; SED, secondary electron detector; TED, transmitted electron detector; SG, scan-current generator; CS, cathodic screen; /, magnified image.

images are more difficult to obtain than in photonic confocal microscopy because the depth of field is too great. (b) The scanning electron microscopes (SEM and STEM) A diagram of a scanning electron microscope is presented in Fig. 4. The electron beam is as well focused as possible, yielding a spot which is displaced over the surface of the sample by magnetic deflection coils (or electrostatic plates) mounted above the sample. The power supply for the coils is also used to move the spot displayed on the oscilloscope or TV screen. The image is acquired point by point, or more precisely line by line. However, as in the case of TEM images, the contrast is determined by intensity differences from one point to another. These intensities are modulated on the TV screen by the electron detector. Three kinds of electron can be used to form an image, namely: (i) Backscattered electrons, which are usually detected by a semiconductor placed around the optical axis and under the pole piece of the last condenser. The image is that of the surface of the sample. The contrast is produced.

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at least in part, by the differences in effective atomic number from one point to another. Such images are both analytical and morphological. (ii) Secondary electrons are collected by a 'nose', a small magnetic element which attracts these relatively low-energy electrons towards a scintillator. The scintillator, generally a luminescent plastic material, emits photons which are guided by a light pipe or optical fiber to the cathode of a photomultiplier. The output of the photomultipHer is connected to the TV screen in the analog mode or to a computer in the digital mode (see below). These two kinds of scanning image are produced from the surface of a sample. They are obtained by surface-scanning electron microscopy (SSEM), or often called simply scanning electron microscopy (SEM). They can accept any kind of sample, small or large, thick or thin, which are prepared by metallization or not, as very low voltages are employed. (iii) Transmitted electrons, from thin samples only, are collected under the sample by a small device, generally a semiconductor, such as that employed in the detection of backscattered electrons. As in surface imaging, the electronic spot is scanned over the sample surface. These transmission electron microscopes, which have been more recently developed than the TEM, are called scanning transmission electron microscopes (STEM). Although simpler than a TEM, such scanning microscopes do not enjoy widespread use in spite of their lower cost. Not all manufacturers provide with a SEM the accessory needed for STEM. Indeed, the question of the better system between TEM and STEM is still debated. It seems that, at high magnification, i.e. high resolution, classical TEM yields better images. At lower magnifications, e.g. 1000 to 50 000, the quality is in competition with the advantages of STEM, namely simphcity, lower cost and the possibility of making surface observations.

(c) Analog and digital images In addition to the direct observation of the screen, the more commonly used medium for the acquisition of analog images is the photographic emulsion. The appropriate size of the silver grains in the emulsion is determined by the resolution and magnification of the image. The image is correct if the diameter of the grains is not more than one-half the resolution at the magnification used; beyond this limit the 'grain' is visible. For example, consider a TEM with 2.5 nm lateral resolution. To obtain a 'good' image for the eye, the higher magnification is 100 000 and the biggest grain suitable for direct observation is 125 fxm in diameter. Of course if the photograph is magnified (as for a slide), the grains must be much smaller. A digital image can be represented as a regular, two-dimensional lattice in which each square represents a pixel. For direct observation, the maximum size of the pixel is 0.25 mm; beyond this limit, the individual pixels become

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visible. For microscope images, the principle is the same, taking into account the resolution and the magnification, as for analog images. In practice, these conditions require a large number of pixels. For each pixel, the number of different intensities - the channel dynamics - can be 2 (black or white) or any greater multiple, e.g. 4, 8, 16, . . . . A digital image is thus very byte-consuming and, until recently, few computers had sufficient memory to store and process it. But now, even a PC may have enough memory to store images with a sufficient number of pixels; thus, numerical imaging has become a very attractive technique, with stimulating possibilities. Besides a greater dynamic range and a better linearity in the acquisition of each pixel, contrast enhancement, comparison of images, zoom effects, quantification of any point, are among the more interesting possibilities offered by image processing. Before considering the problem of coupling an electron microscope with a Raman microprobe, it should be noted that the smallest photonic probe in the UV region (in vacuum), is approximately 0.25 |xm, as determined by the diffraction limit. Thus, in this application high magnification is not required. In practice, magnifications up to 10 000 are quite sufficient.

III. COUPLING OF RAMAN AND ELECTRON MICROSCOPIES A. General Considerations To carry out molecular analyses by Raman spectroscopy in an electron microscope, i.e. from submicroscopic areas, it is necessary to integrate the electron microscope with an optical system which focuses the laser beam and collects the scattered photons. Since the magnetic fields of the electron optical system have no influence on the photons, the problems to be encountered are primarily mechanical. Because spherical aberrations in the electron microscope cannot be corrected, the angular aperture of the electron lenses is very small, i.e. the focus is generally long. Thus, it would seem to be easy to design magnetic lenses for an instrument adapted to include a photon optical system. On the other hand, it is generally difficult to place a Raman microscope within an electron column which was not designed for that purpose. Focusing the laser is necessary in order to obtain good optical quality, but high numerical apertures are required only if the focal spot must be as small as possible. To obtain a good, circular laser probe, the best design is to mount the photon optical system coaxially with the electron microscope. To yield good efficiency, the optical system for collecting the Ramandiffused photons is necessarily of large numerical aperture. It must be remembered that the sampled volume in electron microscopy is smaller than

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Figure 5 Principle of a 47r-steradian, bi-ellipsoidal collector. EB, electron beam; S, sample; EMi and EM2, ellipsoidal mirrors; M, mirrors; OLi and OL2, objective lenses; BS, beamsplitter; L, laser; RS, Raman spectrometer.

that investigated with photon imaging. The best collecting system, from this point of view, would be spherical - a 47r-steradian optical combination such as represented in Fig. 5 (Delhaye and Truchet, 1987a). However, an optical system with this configuration is not easy to adjust within the electron microscope column. Furthermore, the sample holder, as well as the sample itself, creates a spatial anisotropy which generates optical aberrations, and thus further perturbs the system, if it is also used to focus the laser. In general, this configuration is not compatible with the classical electron microscope because the sample is placed inside the electromagnetic objective lens, leaving very Httle space below it. On the other hand, the focus of the last condenser is generally long enough to place a device above the objective. This simpler configuration, which permits collection within the 2TT steradians above the object plane, will be considered below. Figure 6 shows the collection efficiency and the numerical aperture of the objective as functions of the aperture half-angle. The collection efficiency (CE) is defined as the percentage of the volume of the upper half of a sphere centered on the focus at the surface of the sample.

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0.75

0.50f-

0.25

20

30 40 50 60 70 Aperture half-angle (degrees)

80

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Figure 6 Collection efficiency and numerical aperture as functions of the aperture half-angle. The collection efficiency is defined as the percentage of the volume of the upper half of a sphere centered on the focus at the surface of the sample.

B. Photon Optics Collection

It is possible to drill a hole for the electron beam at the center of a classical photonic objective and to place it in the last magnetic lens (condenser) of the illuminating part of the electron microscope. However, this is not a suitable solution because the central hole introduces specific aberrations. Besides, the glass is contaminated by backscattered electrons; the shorter the working distance (the greater the numerical aperture), the greater the contamination. It is far more practical to use mirror optics. The holes for the electrons are easier to drill and the distances between the reflecting surfaces and the sample are, in almost all cases, greater; thus, the possible contamination

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Figure 7 Schematic diagram of a Cassegrain-Schwarzschild-type mirror objective in the last condenser of an electron microscope. S, sample; ML, magnetic lens; CSMO, Cassegrain-Schwarzschild mirror objective; M, mirror; EB, electron beam; HLC, hollow light cyhnder; W, window; BS, beamsplitter; L, laser; RS, Raman spectrometer. by backscattered electrons is reduced. Finally, this system is rigorously achromatic. The wavelength of laser excitation may thus be moved easily from the UV to the near-IR with the same optical arrangement without reahgnment. Various instruments of this type have been developed or may be created; some important examples are illustrated in Figs 7-9. Double-mirror objectives were adapted from the system of Cassegrain, although their characteristics were extensively studied by Schwarzschild. Commercially available, this kind of mirror objective was generally designed with low numerical apertures, e.g. 0.2 to 0.65, depending on the manufacturer, as the surfaces were spherical. However, with the development of digital machines, such as that of Moore, aspherical surfaces have now been fabricated. A mirror objective with a 0.9 numerical aperture and a 0.5 fxm lateral resolution was perfected a few years ago by the companies Rheosc and Optics-for-Research (France). Unfortunately, the very critical mechanical adjustment of this optical system could not be made. Among the characteristics of the double-mirror objective, the most interesting in the present application is the existence of a central obscuration. Thus, a hole for the electron beam can be made in the corresponding part

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Figure 8 Schematic diagram of a completely achromatic optical collection system which employs ellipsoidal mirrors above the sample for coupling the Raman laser probe to the electron microscope. S, sample; EB, electron beam; EMi, first ellipsoidal mirror with a large numerical aperture (0.999); PM^, plane mirror used as sample holder; EM2, second ellipsoidal mirror (N.A. converter); PM2, plane mirror; CSMO, Cassegrain-Schwarzschild mirror objective. of a mirror without any modification of its optical properties (Fig. 7). These Cassegrain-Schwarzschild objectives are very suitable for coupling a photonic optical system with an electronic one. As early as 1958, Castaing and Deschamps (1958), at the ONERA,* near Paris, used this system for their electron probe. Its main disadvantage is, however, its small numerical aperture, which makes it a relatively inefficient Raman laser probe. Collecting light in an electron probe for cathode luminescence has long been a problem, and various other mirror optical systems were designed by * Office National d'Etudes et de Recherches Aerospatiales.

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Figure 9 Schematic diagram of a completely achromatic optical collection system which employs parabolic mirrors above the sample for couphng the Raman laser probe to the electron microscope. S, sample; EB, electron beam; PMi, collecting parabolic mirror, with a hole for the passage of the electron beam; PM2, transfer parabolic mirror (without hole); G, glass lens system; BS, beamsplitter; L, laser; RS, Raman spectrometer. different researchers (Giles, 1975). Off-axis ellipsoidal or parabolic mirrors were commonly used, with relatively good efficiency. Recently, the company Hitachi presented a system for cathode luminescence which is claimed to yield an efficiency of 90% (see Fig. 6). However, such systems are not satisfactory for laser Raman analysis. To focus the laser beam correctly, optical systems with circular symmetry are required, thus ruling out any off-axis system. The parabolic or ellipsoidal mirror must be mounted coaxially with the electron optical system. For such mirrors, and for a small and thin sample, numerical apertures as great as 0.999 can easily be achieved, which is, at the present time, far better than that obtained with the best objective lenses.

C. Transfer Optics

1. Column

Output

To transfer the light collected by a coaxial system and to conduct the laser Hght to it, two cases are to be considered:

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(i) If the sample is placed in a classical TEM, the whole photonic system has to be placed above the sample, in the illuminating part of the column. (ii) The light can be directed beneath the sample only with the STEM or SEM instruments. If the photonic objective is of the Cassegrain type, a plane mirror and a window allow the rays to be deflected out of the column. A beamspHtter and a set of adapted achromatic lenses can be employed to adjust the optical system to both the laser and the Raman spectrometer. In the case of an ellipsoidal objective, a plane mirror is used as the sample holder to reflect the light to the upper part of the column (Delhaye and Truchet, 1987b). However, in this case, the numerical aperture of the reflected light is too large to direct it outside the column, even with a Cassegrain objective. It is then necessary to design a second ellipsoidal stage (Fig. 8). This device, which is very difficult to adjust, has still not been tested experimentally. Another solution, which is mechanically much simpler, is to use directly a parabolic mirror whose axis is perpendicular to the axis of the electron column. The axial symmetry, which is not respected in this case, is approximately achieved with the aid of a second parabolic mirror, identical to the first, but in opposition to it (Fig. 9). The wave surface, which is initially perturbed after reflection by the first mirror, is then realigned. This optical arrangement has not yet been evaluated. When the space under the sample is sufficient, a very simple system is to place a classical objective at the second focus of the eUipsoidal mirror. At the output of this objective, the rays are approximately parallel. Such a setup, which has been developed, requires small samples, as shown in Fig. 10. The shadow of a sample which is too large reduces the numerical aperture of the ellipsoidal mirror, independent of the position of its pupil, i.e. its edge. This arrangement is only well adapted to STEM, in which samples must be small and thin. However, the collecting system for transmitted electrons must be small enough not to create shadows. With bulk samples, such as for SEM, it is suitable if the sample size is in the mm range. The problem of the electron imaging mode then arises. A simple hole at the apex of the ellipsoidal mirror allows the backscattered electrons to be collected, but the mirror itself prevents the collection of secondary electrons. In summary, wide numerical aperture Raman systems are not easily compatible with SEM imaging in the secondary electron mode. For this application, Cassegrain objectives are more suitable. However, highefficiency laser Raman probes are compatible with SEM in the backscattering mode and with STEM equipped with a small collector of transmitted electrons.

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EB

Figure 10 Schematic diagram of a very efficient collection system which employs eUipsoidal mirrors and glass objectives for coupHng the laser Raman probe to the electron microscope. EB, electron beam; PP, polar pieces (last electromagnetic condenser); BED, backscattered electron detector; EM, ellipsoidal mirror (0.999 N.A.); 5", sample; OL, objective lens; M, plane mirror; W, window (for vacuum); BS, beamsplitter; L, laser; RS, Raman spectrometer.

2. Column, Laser and Raman Spectrometer

Connection

To connect these units, the simplest solution would be to attach the laser and the spectrometer to the column and to use plane mirrors to direct the hght beams. In fact, this solution is not suitable in practice, as excessive mass loading and optical misalignment can make it unreHable. To place the laser and the spectrometer on a frame separated from the column is no more reUable because, in almost all cases, the columns are mounted on rubber blocks. The use of optical fibers is, then, preferable, despite the specific problems which they may introduce. For use with a laser the optical fiber must be of the monomode type, although the direction of the polarization vector may not be well defined. At the output of the laser, a specific device has been developed to focus the laser beam into the optical fiber (Fig. 11). With this system powers as high

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EMP

Figure 11 Schematic diagram of the prototype of the Raman-Castaing microprobe. Left side: EM?, electron microprobe (elemental analysis); EC, electron column; XS, X-ray spectrometer. Right side: RMP, Raman microprobe (molecular analysis); C, Cassegrain-Schwarzschild mirror objective (observation and analysis); W, window (vacuum); BS, beamsplitter; RM, retractable mirror; O, ocular (eyepiece for observation of the sample surface); Fj, interference filter; F2, notch filter; Dj to D4, devices for connection with optical fibers; OFi, monomode optical fiber; OF2, multimode optical fiber; L, laser; RS, Raman spectrometer. as 1 W can be transmitted, although the manufacturer indicates that the fiber may be destroyed due to stimulated Brillouin scattering above 100 mW. At the output of the optical fiber a simple- or double-lens system has been developed for optical combination with the Cassegrain or ellipsoidal objective (Fig. 11). Specific devices have been created to focus the Raman light collected by the objective on the entrance of a multimode optical fiber which transmits it to the spectrometer. The Raman spectrum of the optical fiber is very intense in both multimode and monomode propagation. Thus, these two optical fibers produce spurious light which must be suppressed. For monomode transmission in a fiber, the interference filter commonly used in Raman spectroscopy to eliminate the plasma lines of the laser is placed between the fiber and the objective in order to eliminate its Raman spectrum. In multimode transmission in a fiber the rejection of its Raman spectrum is achieved by using notch filters. By eliminating the Rayleigh diffusion and the specular laser light, these new filters eliminate the main cause of fiber spectrum excitation and make possible

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the use of optical fibers to couple Raman spectrometry with electron microscopy. D. Analysis Conditions

1. Efficiency With a SEM or STEM equipped with a 0.999 N.A. optical system, the efficiency is better than that of a conventional Raman microprobe. At present the best lens objectives have a numerical aperture of 0.95. Figure 6 indicates that the efficiency is 69% at an N.A. of 0.95 and 96.5% at 0.999 N.A. With a Cassegrain objective, the highest numerical aperture is less than 0.65, which yields an efficiency of 20%, taking into account the apical obscuration. In this case, the Raman laser probe has the same detectivity as a conventional one equipped with a 20x objective. 2. Samples Under Vacuum In a conventional microscope, the sample is in air or immersed in water or oil. In addition to the enhancement of N.A., the use of hquids protects the sample against heating and subsequent damage. Air also provides some sample coohng, as well as allowing living cells to be studied. In electron microscopy, the sample is in vacuum; thus, its resistance to heating by the focused laser beam is somewhat reduced. The method of sample holding is important, as a thin section on a grid for electron microscopy is especially fragile, whereas a bulk sample is more resistant to laser radiation. It is important to keep this serious problem in mind for Raman studies of a sample in an electron microscope. 3. Sample Size and Analytical Efficiency With the electron microscope the Raman analyzed volume is the same as that in conventional Raman spectroscopy, although such samples can be observed only in SEM. Even with a large numerical aperture the depth of analysis is at least 2 iJim (Dhamelincourt et al., 1991). In TEM or STEM the sample must be less than O.liuim thick. The corresponding loss in Raman intensity is by a factor of 20. Under such conditions only samples with very large scattering cross-sections can be studied. Moreover, electron optics are used to image very small areas; thus, the laser focal point should be as small as possible. It would seem, then, that UV lasers would be more suitable sources than red lasers. On the other hand recent results in the reduction of fluorescence have been obtained with NIR

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laser light (see Chapter 3). It is also possible to increase the power of the laser excitation, but the resistance of the sample in vacuum may be seriously Hmited. This criterion is especially important in the UV region, as well as in the NIR. E. Conclusion To obtain vibrational spectra from samples observed with an electron microscope, it is necessary to design photonic devices that are compatible with the electron optical system. Thus, mirror objectives are more suitable than lenses. Two kinds of mirror objectives have been employed, Cassegrain and ellipsoidal. The Cassegrain introduces a mechanical problem, as the last condenser lens has to be designed especially to house it. On the other hand, Cassegrain objectives do not disturb the imaging system in SEM, TEM or STEM. Its use results in a relatively weak Raman microprobe but, as pointed out above, in SEM any kind of sample can be studied. With the ellipsoidal mirror, only small samples, thin or bulk, can be analyzed, but with better Raman performance. The electron imaging is not disturbed in TEM or STEM, only slightly perturbed in SEM when it is employed in the backscattering mode and very perturbed in the secondary electron mode of SEM.

IV. X-RAY AND RAMAN MICROSCOPY COUPLING A. Fundamental Principles (Benoit et a/., 1987) There are two kinds of stimulated X-ray emitted under electron bombardment. (i) As previously described, X-rays are emitted at each inelastic scattering within the sample; this emission is the Bremstrahlung. It is a noncharacteristic emission which contributes to the background noise. (ii) An impinging electron may eject an electron from an inner shell of an atom (K, L or M); then, during de-excitation of the atom, X-rays are emitted. Their energies (or wavelengths) are characteristic of the chemical nature of the atom. Chemical elements are defined by Z, their atomic number; the energy or the electron of a given layer, K(ls) for example, is clearly dependent upon the charge of the nucleus, i.e. Z, which determines the chemical nature of the atom. The spectrometry of X-rays emitted under electron bombardment is a powerful method for elementary chemical analysis. It is often referred to as

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EDS

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WDS

Figure 12 Schematic diagram of the two principal X-ray spectrometers. EB, electron beam; RX, X-rays; S, sample. Left side: energy-dispersive spectrometer (EDS); SiLi, detector; C, computer (multichannel digitalization); ES, energy spectrum. Right side: wavelength-dispersive spectrometer (WDS) of the Castaing microprobe; BC, Bragg crystal; D, detector; C, computer (mechanical drive and data treatment); MS, monochannel spectrum.

electron probe microanalysis (EPMA). Actually, this method is punctual, as it is implemented with a focused electron beam (10 nm-10 |xm) and it is nondestructive. Two techniques are available for the analysis of the X-ray emission (Fig. 12). (i) First, Castaing (1951) developed a mechanically complex but very efficient system which was adapted from the classical X-ray spectrometers of Johann and/or Johanson (Ruste, 1975). In these systems X-rays are counted with a Geiger-Miiller-type detector, but adjusted to proportional counting. This system is named wavelength dispersive spectrometry, or WDS. Its main advantage is a very good specificity. The energy resolution is currently 10 eV or less. Quantitative analysis is somewhat complicated, but is now well known and reUable. The limit of detectivity is controversial, but it has been estimated to be as low as 5 x 10~^^g for iron in biological samples (Quintana, 1980). (ii) A semiconductor (SiLi) coupled to a multichannel electronic system (now a computer) produces a current which is proportional to the energy

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of the X-rays. This system is named energy dispersive spectrometry, or EDS. The entire X-ray beam is displayed on a screen for a given acquisition time (often 100-300 s), which is more rapid and easier than WDS. On the other hand, the energy resolution is Hmited, typically to 150 eV. It is less specific than WDS; quantitative analysis is identical and lower limits of detectivity similar. The EDS systems are less expensive than WDS, which explains their relative success. Numerous SEM instruments are now equipped with a diode for EDS analysis. On the other hand, Castaing machines are regarded as more sophisticated analytical tools. The above considerations demonstrate the complementarity of Raman and EPMA methods. The application of these techniques in the same instrument and at the same point in the sample allows a complete chemical analysis, i.e. elemental, molecular and structural, to be obtained. More than a combined instrument, the Raman-Castaing microprobe is a new analytical tool. Moreover, as the column is the same as in an electron microscope, the coupling of Raman analysis and electron imaging considered previously is perfectly feasible. The similarity between electron microscopy and electron probe analysis has been known for a long time, and throughout the world numerous manufacturers have built and sold SEM, TEM and STEM systems equipped with a diode for EDS or with a Castaing device for WDS, or both. For the reasons summarized above, the conditions for coupling laser Raman microscopy with EPMA are similar to those required for its coupHng to the electron microscope. In practice, the best system is obtained by coupling the Raman probe to an electron microscope equipped for X-ray analysis.

B. Coupling Conditions (Truchet and Delhaye, 1988) The Cameca electron microprobes are built with a Cassegrain objective mounted in the pole pieces of the last condenser. They are pre-adapted to coupling with a Raman microprobe without any disturbance of the working electron probe, but with relatively poor Raman sensitivity. For higher Raman efficiency the ellipsoidal system is preferred to the Cassegrain configuration. It is employed in the CAMECA instrument, as well as in others in which the distance between the pole pieces of the last condenser and the sample is at least equal to 9 mm. If the electron probe is mounted as for SEM, the ellipsoid is combined with the lenses objective, as previously described. When the electron microprobe is associated with TEM, the use of a plane mirror as a sample holder is required. Furthermore, the upper part of the column has to be modified to house the photon optical system. This modification can also be made for the Cassegrain configuration.

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Figure 12 shows the resulting Cassegrain configuration for X-ray analysis. The Cassegrain configuration has no effect on the analytical function of the electron microprobe. On the other hand, the surrounding structure of a high numerical aperture system, such as an eUipsoidal mirror, absorbs the X-rays. This problem is easily solved by driUing a small hole in the mirror, just in front of the X-ray spectrometer, whatever the nature of the analytical system. For EDS, the diameter of the hole is made sUghtly greater than for WDS (2-2.5 mm versus 1-1.5 mm). Experience with WDS has clearly demonstrated that these holes have no influence on the X-ray beam and, therefore, on the results of elemental analysis. These arguments do not apply to the probes themselves. A given point of a sample which is simultaneously bombarded with electrons and photons may be destroyed, whereas the same energy and/or power of each beam separately does not necessarily have this effect. Another aspect to be considered is the so-called contamination spot, which is well known to electron probe specialists. It consists of a layer of burned oil (usually from vacuum pumps), which absorbs both the laser beam and the Raman-scattered photons. Clean-vacuum and anticontamination systems are required to avoid this problem. If these are not available, it is essential to make the Raman analysis before the electron probe investigation. This author (M. Truchet) has never observed any contamination spot with the laser beam, even at high power.

C. Applications

Whatever the type of sample, the domain of application of a coupled microprobe is general when elemental and molecular and/or structural analyses are required at a given point. If the point is smaller in size than 1 |xm, electron imaging is also required - from the surface or by transmission, depending on the sample and its thickness. The analyzed volume is in the range 0.01-10 |xm-^, i.e. masses of IQ~^^-1Q~^^ g. Such masses are currently analyzed by electron microprobes but, for Raman analysis, masses of 10"^^ g are more suitable, except for substances having very high scattering cross-sections. Such a requirement of elemental and molecular analysis is potentially valuable in any field of research and/or production control, e.g. biology, mineralogy, metallurgy, materials science, semiconductor and ceramics technology, etc. At the present time, the only existing instrument of this type appears to be that developed in this laboratory.* Thus, the applications are still limited, although the results already obtained with

*Laboratoire d'Histophisologie Fondamentale, Universite de Pierre Marie Curie, 12, rue Cuvier, 75005 Paris, France.

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samples as varied as histological sections, iron sheets or white pigments are promising (Truchet and Delhaye, 1992) and are auspicious for the future of the coupHng of complementary methods. No specific difficulties have been encountered, except the reduced resistance of the sample under vacuum and the absorption by the contamination spot, which have already been described. An unexpected observation has recently been made concerning sample fluorescence. It is well known by Raman spectroscopists that this parasitic light can often be reduced by sample 'burning' - irradiation over an extended time prior to the recording of the Raman spectrum. The resulting decrease in fluorescence is also useful in Raman microspectrometry, although it is time-consuming. It has been observed that this phenomenon does not occur for some samples observed under vacuum. This result suggests that the photolytic decomposition of the fluorescent substance requires air, i.e. oxygen (Truchet et al., unpublished).

V. COUPLING OF SECONDARY ION MASS AND RAMAN SPECTROSCOPIES A. Introduction Although it is widely used in industry and research for the analysis of semiconductors, as well as in metallurgy, secondary ion mass spectroscopy (SIMS) is not as prevalent in geology or biology, despite its analytical power. However, the coupling of this method with Raman microscopy may offer interesting possibihties. Secondary ion emission is an elemental analytical method. As an electron microprobe, it can be used to identify chemical elements. Furthermore, compared with EPMA, it has some specific advantages. First, the method, which is based on mass spectrometry, is sensitive to isotopes in either natural or artificial abundance. Second, light elements, which are very difficult to detect in EPMA, are easily identified in SIMS. Third, for numerous elements (especially light elements, alkaline, alkahne-earth, halogens and some metals such as Al) the mass spectrometric method is much more sensitive. On the other hand, it is not quantitative because a model of the sputtering process has not yet been established. Thus, the fluctuations currently observed in concentration and emission intensity are neither predictable nor interpretable. B. Principles of Instrumentation The SIMS can be employed as a simple spectrometer, although it is widely used in the microscope configuration (Castaing and Slodzian, 1962). For a

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MS

DT

- • /

Figure 13 Schematic diagram of a SIMS (secondary ion mass spectrometer) microscope. /G, ion gun; lO, ion-focusing optics; PI, primary ion beam; S, sample; SI, secondary ions (emitted by the sample surface under primary ion-beam bombardment); EL, electrostatic lens (collection and focusing device giving an image of the sample surface with the secondary ions); CO, cross-over diaphragm (entrance slits of the mass spectrometer); MS, mass spectrometer (90° magnetic sector for mass filtering); MFI, mass-filtered image (secondary ions); SS, selecting slits (to select one image); DT, data treatment; /, secondary ion images of the sample surface; S, mass spectra for elemental isotopic analysis. long time, Cameca was the international leader in commercial instruments of this type. More recently, systems manufactured by Charles Evans and Perkin-Elmer have become attractive. In these instruments the imaging is obtained directly w^ith secondary ions and by simultaneous acquisition from all of the pixels or by scanning of the primary ion beam. Figure 13 presents the schematic representation of the first ion microscope (Slodzian, 1964). The general principle, implemented in all of these instruments, involves a primary ion beam (oxygen, argon, or any ion, such as WFs^), generated by a plasmatron or by Hquid-metal guns (Ga, Cs). Under this bombardment, the surface of the sample is sputtered off, emitting its atoms, or clusters of atoms, among which some have lost or captured one or several electrons; these are the secondary ions, both positive and negative. An electric field attracts these ions and an electronic lens focuses them on the entrance slit of a mass spectrometer. The spectrometer can be of the quadrupole type with a magnetic sector - with or without an electric sector - or a time-of-flight instrument (TOP).

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In the microscope configuration, the collecting and focusing electrodes are mounted as an electrostatic lens, yielding an image of the bombarded area of the sample. The spectrometer is then designed to preserve this image, filtered in mass and representative of the spatial distribution of a given ion at the surface of the sample. The lateral resolution of the ion image is approximately 0.5 |xm in the simultaneous mode and can reach a spatial resolution of 0.05 |xm (50 nm) in the scanning mode, especially when liquid-metal guns are employed. Following the spectrometric configurations, secondary ions from mass 1 to mass 250 are detected and localized (magnetic sectors) with a mass resolution of up to several thousand amu (quadrupoles or TOF spectrometers). The mass resolution (M/dM) of these instruments has for a long time been a major problem because of numerous interferences, at a given mass, between elemental, i.e. monatomic ions, which are characteristic of the isotope of a chemical element, and polyatomic ions, which are not. All of the better instruments currently reach 5000-10 000 in mass resolution, which is satisfactory. However, the new Fourier transform mass spectrometer (FT-MS) reaches resolutions as high as 10 million, which opens up a new domain of application of mass spectrometry. Unfortunately, these instruments are not yet available for SIMS.

C. Coupling Conditions

As for the preceding methods, the best optical arrangement is obtained with the use of mirror optics mounted coaxially with the electrostatic lens. In fact, objectives with very long working distances could be used, but their small numerical apertures would result in poor Raman probes. Unlike EPMA or electron columns, Cassegrain objectives are not suitable in this case. Although electrostatic lenses are smaller than magnetic lenses, there is insufficient space to mount a Cassegrain objective. It is thus more suitable to place a parabolic or ellipsoidal mirror around the electrostatic lens. Figure 14 shows an example of this system. In this case, despite the large hole at the apex of the ellipsoid to accommodate the electrostatic lens, the equivalent numerical aperture remains 0.8, which yields a collection efficiency of 50%. If the sample is not too large (i.e. < 1 cm), an objective, placed behind the sample, receives the light collected by the ellipsoid, which is subsequently transferred by it to the spectrometer. The same optical system is suitable for focusing the laser beam. The principal problem encountered in the coupling system described above arises from the contamination of the optical surface by the ions. This difficulty can, in principle, be resolved by placing the ellipsoidal mirror at an electric potential sufficient to repel the ions. However, important modifications must

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Figure 14 Schematic diagram of a Raman microprobe coupled with a SIMS microscope (Raman SIMS). S, sample; si, secondary ions; EL, electric lens; MS, mass spectrometer (for isotopic elemental analysis); EM, ellipsoidal mirror; OL, objective lens; BS, beamsplitter; L, laser; RS, Raman spectrometer (for structural and molecular analysis). then be made in the focusing of the electrostatic lens. Thus far, no experimental work appears to have been done on a system of this type.

D. Applications

Ion sputtering is a destructive method. Therefore, in the case of its coupling with Raman analysis, it would be necessary to perform the vibrational analysis before the mass analysis. This means that the coupling of these two methods has not the same general interest as does the coupUng of Raman microscopy with the electron probe or, more generally, electron microscopy.

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However, in some cases, it would be of fundamental interest to replace the nonisotopic elemental analysis performed by EPMA by an isotopic analysis; SIMS is then required. This method could be employed to localize isotopic labels, such as used in biochemistry, physiology and medicine, as well as in the nuclear industry. Another interesting field of application is the use of ion implantation, especially in semiconductors. In this case, it should be possible to follow the implantation process in real time by Raman spectrometry, and thus to control the crystallinity of the substrate. The applications are analogous in the recent technology of superconductors at relatively high temperatures. Raman analysis is a powerful tool for controlling the epitaxial deposition of the multilayers, although it is not a specific method for characterizing superconductivity. The use of SIMS in mass fragmentometry for molecular analysis is becoming more widespread, especially with the commercialization of new TOF-SIMS instruments. The availability of a true molecular analysis method with the use of this technique would be of considerable interest.

VI. COUPLING OF LASER MASS SPECTROMETRY (LMS, LAMMA, LPMS) WITH THE RAMAN MICROPROBE A. General Considerations This method is similar to SIMS, in that it involves the mass spectrometry of secondary ions. Thus, the general characteristics previously described are the same, i.e. elemental isotopic analysis, high sensitivity for numerous elements, problems of interferences in qualitative analysis and those associated with matrices in quantitative analysis. The main difference between these methods and SIMS is in the nature of the excitation beam. In SIMS, it is a primary ion beam, whereas in laser mass spectrometry (LMS), it is a photon beam. This photon beam is of course a laser beam; but, to produce the micro-plasma which generates secondary ions, a high-power laser is required. It is generally a pulsed neodymium-YAG which results in a discontinuous production of secondary ions. This pulsed mode of operation is fundamental in LMS. With each pulse a part of the sample is destroyed, depending on the energy of the laser pulse (its power and pulse duration) and the characteristic coefficient of energy dissipation of the area around the target. This very important aspect must be considered, since the greater the energy of the laser pulse, the lesser the matrix effects, i.e. the more reliable is the quantitative analysis. However, at high energies the volume destroyed in the sample may reach 100 jxm^, which is incompatible with microscopic analysis. On the other hand, low-energy pulses

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FO

Figure 15 Schematic diagram of the two main modes of laser excitation for micro-mass spectrometry (LAMMA and LPMS). Upper diagram: transmission mode (LAMMA); L, laser; FO, focusing optics; W, window; S, sample; TOF-MS, time-of-flight mass spectrometer; DP, data processing system for isotopic elemental analysis of volumes less than 1 |xm^. Lower diagram: reflection mode (LPMS); L, laser; FO, focusing optics; W, window; S, sample; TOF-MS, time-of-flight mass spectrometer; DP, data processing system for isotopic and quantitative elemental analysis of volumes greater than 1 |xm^. produce very small craters, less than 1 fxm^ in volume. However, at lower energy polyatomic ions are numerous, i.e. qualitative analysis is difficult and matrix effects are so significant that this method has been developed for molecular analysis to identify by the matrix itself. Another important consequence of the discontinuity of secondary ion production is the requirement of special spectrometer systems. In this application the most suitable spectrometer is a TOF. These instruments have been plagued for a long time by insufficient mass resolution. Fifteen years ago, it was less than 200. Now, a mass resolving power of 10 000 is possible, which is sufficient to remove numerous interferences. Another main advantage of TOF is its very wide mass range, i.e. 1 to 10 000.

B. Instrumental Configurations

Two main configurations have been developed for LMS (Fig. 15). In the first, the surface of a bulk sample is sputtered off by the use of a very long working distance photonic optical system; the secondary ions are also collected by

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an electrode above the sample. This configuration, now known as surfaceLMS, was that of the laser probe mass spectrometer (LPMS) of the CEA ('Commissariat a I'Energie Atomique') in France. With this instrument, very high powers are possible. The conditions for reliable quantitative analysis were defined by Eloy (1980). In the second case, the sample is thin. The laser pulse is applied to one side with the use of a very high numerical aperture objective which creates a small hole. The secondary ions are collected from the other side. This configuration was developed in Germany (Hillenkamp et al., 1975) and commercialized under the name of LAMMA by Leybold-Heraeus. Holes as small as 0.5 |xm were routinely obtained with such instruments and numerous studies have demonstrated an impressive lower limit of detectivity of 10"^^g (Hillenkamp and Kaufmann, 1981). Unfortunately, this method is not widespread because of the insufficient mass resolution of the TOP spectrometer. In the meantime, SIMS was developed.

C. Coupling Conditions Because of the optical focusing system of the laser, there are no particular difficulties in mounting a Raman microspectrometer in an LMS microscope. Moreover, in the recent LAMMA instruments, a second laser is employed to localize the position of the pulsed, high-power UV laser. It may also be very easily used as excitation for Raman scattering. The high numerical aperture of the LAMMA optical system should produce efficient Raman probes without any modification. From this point of view, the German instruments are pre-adapted to the present application. However, no experiments appear to have been carried out on this type of instrument.

D. Applications The coupling of a Raman spectrometer to an LMS microscope is easier to make than to a SIMS instrument, although the applications of these two systems are very similar. The two microscopes are equally suited for isotopic analysis and, generally speaking, they have better detectivity than EPMA, although in SIMS some elements such as As, Cd or Hg have such poor ionic yields that EPMA may be more sensitive. However, it has been shown that good results can be obtained on these elements with the use of LMS, although the advantage of ionic implantation offered by SIMS does not exist in LMS. These considerations suggest that an interesting field of application would be to complement the molecular analysis by LMS at low energy with Raman microscopy.

Raman Microscopy and Other Local Analysis Techniques 231 VII. SUMMARY OF COUPLING WITH ELECTRON, ION AND X-RAY MICROSCOPIES

The main problem now encountered by the speciahsts in microsample analysis is the multiplicity of instruments. Clearly, the cost of separate microscopes is a serious consideration. Furthermore, it is often difficult to relocate the same small region in a sample with change in instrument. Thus the use of several different physical methods within the same microscope system would appear to be advantageous. Electron microscopy and electron probe microanalysis have been combined for some time. Now, from an analysis of the coupling conditions, the adjunction of a Raman laser probe appears to be feasible. However, the specific characteristics of mass spectrometry are also interesting, especially for isotopic analysis. It is therefore suggested that a multimode analytical microscope be developed which employs electron imaging by scanning - both surface and transmission - electron probe analysis, Raman microscopy and mass spectrometry - by both primary ion and laser beam excitation. The experience obtained in this laboratory with the couphng of the Raman microscope to the electron microscope and the microprobe has demonstrated that the multimode analytical microscope proposed here is possible and that it would represent an invaluable analytical technique.

VIII, MICROCHROMATOGRAPHY AND OTHER SEPARATION METHODS A. Thin-layer Chromatography

Among various separation techniques, thin-layer chromatography (TLC) is an inexpensive and easy-to-handle method which is useful in the biochemistry and medical fields. After elution by a suitable solvent, the identification of the separated species is mainly performed with the use of Rf values and chemical methods. Conventional spectrometric methods, which are either poorly specific (UV-visible) or limited by lack of sensitivity (infrared and Raman) require further extraction of the analyte from the layer. The spatial resolution obtainable by the Raman microprobe technique appears to be suitable for the direct identification of the substances separated by TLC. However, it is very important to optimize the experimental conditions in order to prevent heating effects and photochemical alteration of the molecule under investigation. For a colored sample, the enhancement obtained by the resonance Raman effect can increase the sensitivity of the method by many orders of magnitude. However, the significant absorption of the exciting radiation can easily

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destroy the colored substance. Photochemical processes are often involved; the relevant parameters are the irradiance, the absorption cross-section and the quantum yield of phototransformation. For carotenoids spotted on sihca gel plates, the photo-oxidation in air has been estabhshed. In this case, flowing nitrogen over the investigated spot during analysis prevents the fading of the molecule (Merlin and Delhaye, 1987). A significant decrease of the temperature of the sample can also minimize the degradation rate (Barry and Mathies, 1982). A well-known technique to reduce the irradiance consists of increasing the irradiated area of the chromatogram spot along a hne optically conjugated with the entrance slit of the spectrometer (Koningstein and Gachter, 1973) with the use of a cylindrical lens or deflection of the laser beam. The scanning device which is available in the new generation of Raman microprobe can be used for this purpose. The deflection of the laser spot on the chromatographic plate, together with rapid recording at low laser power and low temperature, can reduce appreciably the heating and photochemical damage. For colorless samples the Raman signal that is obtained directly from a chromatographic spot, requires a significant concentration of matter; it is thus not a powerful method. Because surface-enhanced Raman scattering (SERS) provides an increase of sensitivity as a result of the large enhancement effect (up to a factor of 10^), it would seem to be an ideal tool for trace analysis and chromatographic detection (see Chapter 9). After a separation on a TLC plate, a colloidal silver suspension is sprayed onto the plate, and SERS spectra with good signal-to-noise ratio are then recorded from the colored spots that are formed. In this way spectra of an analyte molecule in the pg range can be obtained (KogHn and Planar, 1989). However, in addition to a number of practical difficulties (inhomogeneous spraying), there are several fundamental limitations related to the propensity for the molecule to adsorb from the plate onto the metal (Garrell, 1989). SERS can also be used as an in situ detection method to identify analytes separated by gel electrophoresis and capillary-zone electrophoresis; the sensitivity is of the same order of magnitude as that obtained with laser-induced fluorescence spectroscopy (Cheng and Dovichi, 1988) but the spectral information is much more detailed. Recently, Fourier transform Raman spectroscopy has been combined with SERS for the in situ identification of TLC spots without perturbing the fluorescence effect (Lehner et al., 1994). The spatial resolution of the Raman microprobe technique allows the intensity profile of a chemical species in a heterogeneous sample to be recorded. For a macroscopic area, the deflection of the laser beam and the analysis at a characteristic wavenumber can be used for such purposes. For a macroscopic area, the sample must be moved under the microscope objective. This procedure can be applied in the recording of a Raman chromatogram from a TLC plate after elution. A Raman chromatogram recorded from a silica gel plate after the elution of a mixture of carotenoids is shown in Fig. 16.

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OH

5

10

15

20

DISTANCE (mm)

Figure 16 Raman chromatogram of the carotenoids extracted from the sea mussel Mytilus edulis. The acetonic solution of the extract is spotted on a silica gel plate and eluted with a mixture of dichloromethane and ethylacetate (7:3). The plate is moved under the microscope objective at a rate of 10 ixm s~^ (Excitation: 514.5 nm; laser power: 8 mW). The chromatographic peaks observed at 1530 cm~^ (indicated by arrows) correspond to carotenoids; those at 1650 cm~^ are due to fluorescent impurities.

In this application the spectrometer is fixed at a characteristic wavenumber of the stretching mode of the polyenic chain (v ~ 1530 cm~^). The elution profile recorded for a noncharacteristic wavenumber of carotenoid indicates the position of fluorescent contaminants (Lafage-Szydlowski, 1984).

B. High-performance Liquid Chromatography

High-performance liquid chromatography (HPLC) is one of the most important methods used for the qualitative and quantitative analysis of complex media. UV-visible absorption spectroscopy, fluorescence emission and refractive index changes are routinely employed as detection methods. Commercially available detectors offer an excellent sensitivity with singlewavelength or multiwavelength detection capabilities. Because of the flexible geometry allowed by the laser excitation and the low sample volume requirements, the use of Raman spectroscopy for detection has high potential. Since 1979 numerous Raman detection schemes have been proposed and several advantages have been demonstrated, namely: (i) The method is nondestructive, (ii) The selectivity is great, and (iii) The vibrational spectrum is of sufficient quaUty to provide structural information on the molecular analyte. In principle the Raman method can be applied to a large range of molecules.

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but in practice there are several constraints for each individual investigation. The main limitations in the use of normal Raman spectroscopy is the lack of sensitivity and the Raman spectrum of the mobile phase which can obscure many spectral windows in the solute Raman spectrum. In order to overcome these limitations, several methods have been proposed: resonance Raman (Chapput etal., 1979,1980; D'Orazio and Schimpf, 1981; Iriyama etaL, 1983; Koizumi and Suzuki, 1987; Chen and Morris, 1988; Roqueta et al., 1988; Chong etal., 1992), SERS (Freeman etal, 1988; Ni etal, 1989) and coherent anti-Stokes-Raman (Carreira et al., 1980). A typical HPLC system with an on-line, post-column Raman detection cell is presented in Fig. 17a. A Raman chromatogram can be recorded by single-wavelength monitoring (Fig. 17b). The Raman spectrum of each individual analyte can be obtained off-hne (Ni et al., 1989), under stoppedflow conditions (Chapput et aL, 1979, 1980; Iriyama et al., 1983; Koizumi and Suzuki, 1987; Chen and Morris, 1988; Freeman et al., 1988; Roqueta et al., 1988) or under flowing conditions with a multichannel detection system (D'Orazio and Schimpf, 1981; Todoriki and Hirakawa, 1984; Chong et al., 1992). The design of the flow cell is of particular importance because the volume must be adapted to the size of the column and to the flow rate in order to prevent band broadening. The scattered light and background luminescence often obtained with micro-flow cells can limit the sensitivity. Cells with a volume of only a few nl can be interfaced with micro-packed columns. A sheath-flow cuvette allows a volume as small as 50 fl to be employed (Zarrin and Dovichi, 1985). The use of optical fibers has been proposed (Todoriki and Hirakawa, 1984; Chong et al., 1992). Such a system could provide for on-the-fly acquisition of resonance-Raman spectra with correction for sample absorption (Chong et al., 1992). In the SERS experiment the irreproductibility of hydrosol-preparation procedures and the nohlinearity between the SERS signal and analyte concentration can be overcome by the use of the flow-injection technique (Berthod et al., 1987) in which the Ag solution is added to the chromatographic effluent in a post-column mixing coil (Freeman et al., 1988).

IX. OPTICAL WAVEGUIDES: RAMAN SPECTRA OF FILMS AND ADSORBED SPECIES A. Introduction Thin films are in a certain sense only semi-microsamples, although their thickness may be as small as tens of Angstroms. Their investigation is of considerable practical interest, and has presented a challenge to Raman spectroscopists, as it involves rather special experimental techniques.

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I INJECTOR

HPLC t PUMP SOLVENT

C 18 COLUMN ZORBAX ODS

a

BECKMAN MODEL 112

ARGON LASER RAMAN CELL

(a)

ENTRANCE SLIT

RECORDER

AMPLL

PHOTOMUL

RAMAN SPECTROMETER

5

10

EVOLUTION TIME (MIN) Figure 17 (a) Schematic diagram of the HPLC-Raman system, (b) Chromatogram of carotenoids extracted from blood plasma. The intensity of the 1156 cm~^ hne is recorded against time of elution [Solvent: acetonitrile, ethylacetate, n-deconol (71.9:28:0.1); excitation: 488 nm; laser power: 300mW; slit width: 2 4 c m - i ] .

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In the early days of laser Raman spectroscopy it was shown that it was possible to observe the spectra of thin films (Hendra and Loader, 1967,1968). Useful Raman spectra of even monomolecular films were obtained. Thus, the application of Raman spectroscopy to the study of adsorbed species became possible. A number of different sampling systems for the investigation of thin films have been described. The backscattering configuration, which is normally employed in Raman microspectrometers, is useful for the study of relatively thick films (>5 |xm), particularly at normal incidence. For the analysis of thinner films, a method initially proposed by Greenler and Slager (1973) is often used. In this case the sample is deposited as a thin layer on a metallic surface (e.g. Ag or Ni) and laser excitation is applied at glancing incidence. The Raman-scattered light is observed with a microscope objective. It was shown that the optimum angle of incidence for laser excitation is approximately 70°, while the collection efficiency is maximum at around 60° with respect to the surface normal. These parameters depend somewhat on the optical constants of the metallic support (Greenler, 1966). Under these conditions satisfactory Raman spectra of 50 A (5 nm) deposits of benzoic acid were recorded (Greenler and Slager, 1973). The use of a metallic support for the investigation of thin films has become even more interesting with the discovery of surface-enhanced Raman spectroscopy (SERS). The phenomenon of surface enhancement will be treated in Chapter 9, where its importance in the Raman microscopic analysis in biological systems and medicine is emphasized.

B. Integrated Optics A more sophisticated approach to the investigation of thin films by Raman spectroscopy has developed from the early work of R. Dupeyrat and coworkers (Levy et al., 1974). The method involves an integrated optical system in which the sample film serves as a waveguide or dielectric wall. The basic theory of this method has been reviewed by Rabolt and Swalen (1988). In the initial experiments a thin-film sample of refractive index ^2 was deposited on a support surface. The laser beam was focused on the sample through a coupling prism, as shown in Fig. 18. If the angle of incidence and the polarization direction are properly chosen, the light propagates as a guided wave in the sample film. A microscope objective is employed to collect the scattered light. The image of the entrance sHt of the monochromator is adjusted so that its width is equal to the diameter of the propagating laser beam and its length corresponds to the distance L (Fig. 18). The coupling of the excitation to the waveguide sample is determined by the boundary conditions which are functions of the refractive indices of the

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Laser beam Sample film Prism

///////7/////////////

/

Figure 18 Application of the waveguide technique to Raman studies of films.

media. It was shown by Levy and Dupeyrat (1977) that one or several modes of either the TE (transverse electric) or TM (transverse magnetic) polarizations can propagate in a film of thickness a according to the relation 2kn20-cos 62 - ^12 - ^23 = 27rm,

m = 0,1,2,,

(1)

where k=27r/\o, with AQ equal to the vacuum wavelength of the light, S12 and ^23 are the phase shifts at interfaces 1-2 and 2-3, respectively, and $2 is the angle of reflection within the waveguide (see Fig. 19). The phase shifts depend on the refractive indices rii and ^3, and propagation constant a = n2sin02fora given polarization (TE or TM). For the TE modes, as an example, Eq. (1) yields the family of curves shown in Fig. 20 for a typical case in which rii = 1.46, ^2 = 1.50 and ^3 = 1. For a given value of m, it is seen that if the film thickness a decreases, a approaches rii and the energy penetrates more and more deeply into the support. However, when a increases, a tends towards the value of /t2 ^nd the energy is contained in the waveguide layer. In the latter limit, the field of evanescent or exponentially damped waves becomes equal to zero. It is thus possible to obtain separately or simultaneously the Raman spectrum of the waveguide sample or its support by varying the thickness of the sample layer. The advantages of the waveguide method in Raman spectroscopy were pointed out by Levy and Dupeyrat (1977), who estimated that it could yield an intensity gain of a factor of 2000 with respect to the backscattering geometry under similar sampling conditions. Furthermore, unlike the glancing-incidence geometry, the waveguide technique allows a choice of polarization directions to be made. The TE modes can be excited by polarizing the laser beam in the Z,X plane (see Fig. 19) and choosing the proper angle of incidence. Rotation of the polarization so that the electric vector is parallel to the Y axis, accompanied by a small adjustment of the angle of incidence, results in propagation of a TM mode. It should be noted, however, that there is always a lower limit to the thickness of a film which

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///////y/. Figure 19

112

Analysis of light propagation in a film deposited on a substrate.

-

1.A9

m=0

/

/T7=1

/

/r? = 2

y

m = 3 ^'^^^'^

1.A8 c o

o en

O

Q.

o

1.47

-

^ 1 ""

10 Reduced f i l m thickness ,

a/X.

Figure 20 Propagation constant a = «2 sin 62 as a function of reduced sample thickness for TE modes. In this typical case ni = 1A6, n2 = 1.5^ and ^3 = 1.

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Laser beam Figure 21 The multilayer integrated optical system. Excitation is at an angle 6i with respect to the vertical axis. can be used as a waveguide. In the above example for m = 0, it is equal to 0.56A, as determined by the intersection of that curve with the line a = rii.

In subsequent work by Cipriani et at. (1974) a multilayer structure was developed which can be employed to obtain the Raman spectra of thinner films. The analysis of this system is based on Fig. 21; the four layers are characterized by their respective refractive indices and thicknesses. In this configuration the exciting laser beam enters the system from below through medium 1 at an angle of Si with respect to the vertical (Z) axis. Media 2 and 3 are both thin films, while the semi-infinite medium 4 is usually air. The Raman scattering is observed as before, from above, with a microscope objective. The parameters of the various media are chosen to produce inhomogeneous fields in media 2 and 4, and homogeneous ones in medium 3. For Hi, ^3 and n^ real, the necessary conditions are realized if 6i is larger than the critical angle defined by sin~^(^4/ni). Equation (1) represents the resonant condition in the thin layer (3) if 3^2 and 623 are replaced by 623 and 634, respectively. The phase shift ^23 depends on the presence of medium 1, and, as 0-2 approaches infinity, the system becomes (aside from notation) equivalent to that of Fig. 19. For a typical multilayer system, the energy density in layers 2, 3 and 4, relative to that in layer 1, is represented in Fig. 22. It is apparent that a very large increase in energy density can be obtained in film 2. It is estimated that in this case the Raman scattering is approximately 3000 times greater than that obtained with the use of the backscattering configuration (Levy and Dupeyrat, 1977). Although the scattering intensity is about the same as in the waveguide method described above, the thickness of the film can be significantly reduced (Rabolt et al, 1979). C. Applications

The integrated optical technique outlined above has been developed and applied to the Raman spectroscopic investigation of various thin-layer

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Relative

energy density

Figure 22 Relative energy density as a function of vertical distance in a multilayer system.

systems (Rabolt et al., 1980). It has also been employed to obtain Raman spectra of films of the Langmuir-Blogett type (Barbaczy et al., 1987; Rabe et al., 1987). The spectra are indicative of molecular orientation on surfaces and are sensitive to order-disorder transitions. This technique has been recently combined with Fourier transform Raman spectroscopy to study organic films, polymer laminates and molecular composites (Zimba et al., 1990). In these applications excitation in the near-infrared region with a YAG laser (A = 1.064|xm) was employed in order to eliminate fluorescence interference (see Chapter 3).

REFERENCES Barbaczy, E., Dodge, F. and Rabolt, J. F. (1987). Appl Spectrosc. 41, 176. Barry, B. and Mathies, R. (1982). 7. Cell Biol. 94, 479. Benoit, D., Grillon, F , Maurice, F., Roinel, N., Ruste, J. and Tixier, R. (1987). Microanalyse par sonde electronique: spectrometrie des rayons X. ANRT, Paris. Berthod, A., Laserna, J. J. and Winefordner, J. D. (1987). Appl. Spectrosc. 41, 1137. Carreira, L. A., Rogers, L. B., Gross, L. P., Martin, G. W., Irwin, R. M., Von Wandruska, R. and Berkowitz, D. A. (1980). Chem. Biomed. Environ. Instr. 10, 249.

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Castaing, R. (1951). Doctoral thesis, Conera Paris. Castaing, R. and Deschamps, J. (1958). La Recherche Aeronautique. ONERA, Paris. pp. 41-51. Castaing, R. and Slodzian, G. (1962). / . Microsc. 9, 395-410. Chapput, A., Roussel, B. and Montastier, J. (1979). C. R. Acad. Sci. C289, 283. Chapput, A., Roussel, B. and Montastier, J. (1980). / . Raman Spectrosc. 9, 193. Chen, C. Y. and Morris, M. D. (1988). Appl Spectrosc. 42, 515. Cheng, Y. F. and Dovichi, N. J. (1988). Science 242, 562. Chong, C. K., Mann, C. K. and Vickers, T. (1992). / . Appl Spectrosc. 46, 249. Cipriani, J., Racine, S., Dupeyrat, R., Hasmonay, H., Dupeyrat, M. Levy, Y. and Imbert, C. (1974). Optics Comm. 11, 70. Delhaye, M. and Truchet, M. (1987a). In: R. H. Geiss (ed.), Microbeam Analysis. San Francisco Press, San Francisco, pp. 163-164. Delhaye, M. and Truchet, M. (1987b). Patent 87 09883 (CNRS-ANVAR). Dhamelincourt, P., Delhaye, M., Truchet, M. and Da Silva, E. (1991). / . Raman Spectrosc. 22, 1. D'Orazio, M. and Schimpf, U. (1981). Anal. Chem. 53, 809. Eberhart, J. P. (1976). Methodes physiques d'etude des mineraux et des materiaux solides. Douin, Paris. Eloy, J. F. (1980). Proc. 5th Int. Symp. High Purity Mat. Sci. Techn., DDR Akad. Wiss., Dresden, 1980. Freeman, R. D., Hammaker, R. M., Meloan, C. E. and Fateley, W. G. (1988). Appl. Spectrosc. 42, 456. Garrell, R. L. (1989). Anal. Chem. 61, 401. Giles, P. L. (1975). Cathodoluminescence. In: P. Echlin and P. Galle (eds). Biological Microanalysis. SFME, Paris,.pp. 357-370. Greenler, R. G. (1966). /. Chem. Phys. 44, 310. Greenler, R. G. and Slager, T. L. (1973). Spectrochim. Acta 29A, 193. Hendra, P. J. and Loader, E. J. (1967). Nature 216, 789. Hendra, P. J. and Loader, E. J. (1968). Nature 111, 637. Hillenkamp, F. and Kaufmann, R. (1981). Fresenius Z. Anal. Chem. 308, 1-320. Hillenkamp, F., Kaufmann, R., Nitsche, R. and Unsold, E. (1975). Appl. Phys. 8, 341. Iriyama, K., Ozaki, Y., Hibi, K. and Ikeda, T. (1983). / . Chromatogr. 254, 285. Koglin, E. and Planar, J. (1989). Chromatography 2, 194. Koizumi, H. and Suzuki, Y. (1987). / . High Res. Chromatogr & Chromatogr. Comm. 10, 173. Koningstein, J. A. and Gachter, B. F. (1973). / . Opt. Soc. Am. 63, 892. Lafage-Szydlowski, N. (1984). Thesis 'Docteur Ingenieur', University of Lille. Lehner, C , Sawatzki, J., Koglin, E., Kramer, H. and Kawai, N. T. (1994). In: N. T. Yu and X. Y. Li (eds), Proc. Int. Conf. Raman Spectrosc. The Hong Kong University of Science and Technology, Hong Kong. Additional volume, p. 289. Levy, Y. and Dupeyrat, R. (1977). /. Phys. (Col. C5) 38, 253. Levy, Y., Imbert, C , Ciperiani, J., Racine, S. and Dupeyrat, R. (1974). Optics Comm. 11, 66. Magnan, C. (1961). Traite de Microscopie Electronique, vol. 1. Hermann, Paris. Merlin, J. C. and Delhaye, M. (1987). In: J. Stepanek, P. Anzenbacher and B. Sedlacek (eds). Laser Scattering Spectroscopy of Biological Objects (Studies in Physical and Theoretical Chemistry). Elsevier, Amsterdam, vol. 45, p. 49. Ni, F., Thomas, L. and Cotton, T. M. (1989). Anal. Chem. 61, 888. Quintana, C. (1980) Biol. Cell. 39, 151. Rabe, J. P., Swalen, J. D. and Rabolt, J. F. (1987). / . Chem. Phys. 86, 1601.

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Rabolt, J. F. and Swalen, J. D. (1988). In: R. J. H. Clark and R. E. Hester (eds), Spectroscopy of Surfaces. John Wiley & Sons, Chichester, p. 1. Rabolt, J. R , Santo, R. and Swalen, J. D. (1979). AppL Spectrosc. 33, 549. Rabolt, J. R , Santo, R. and Swalen, J. D. (1980). AppL Spectrosc. 34, 517. Roqueta, C , Merlin, J. C , Fournier, C. and Hecquet, B. (1988). In: R. J. H. Clark and D. A. Long (eds), Proc. Int. Conf. Raman Spectrosc. (London). J. Wiley & Sons, Chichester, p. 717. Ruste, J. (1975). J. Microsc. Biol. Cell. 22, 151-162. Slodzian, G. (1964). Doctoral thesis. University of Paris. Todoriki, H. and Hirakawa, A. Y. (1984). Chem. Pharm. Bull. 32, 193. Truchet, M. and Delhaye, M. (1988). J. Microsc. Spectrosc. Electron. 13, 167175. Truchet, M. and Delhaye, M, (1992). In: W. Kiefer (ed.), Proc. Xlllth Int. Conf Raman Spectrosc, Wurzburg, Germany. John Wiley & Sons, Chichester, pp. 1068-1069. Zarrin, R and Dovichi, N. J. (1985). Anal. Chem. SI, 2690. Zimba, C. G., Hallmark, V. M., Turrell, S., Swalen, J. D. and Rabolt, J. F. (1990). / . Phys.Chem. 94, 939.

Application to IVIaterials Science Paul Dhamelincourt and Shin-ichi Nakashima

1. INTRODUCTION When Raman spectroscopy is applied to the analysis of microscopic particles or microscopic volumes within a heterogeneous sample, it is usually assumed that all of the physical phenomena involved in the Raman scattering of Hght from macroscopic samples are the same. However, it is well known that scattering from samples whose dimensions become comparable to the wavelength of the excitation represents a special case. Calculations based on the Lorenz-Mie formahsm (Kerker, 1969) show that a strong increase in the internal electric field may occur as a result of morphology-dependent resonances when microsamples of well-defined geometries (e.g. spheres, spheroids and cylinders) are illuminated by a plane wave at certain wavelengths. Physically, these resonances result when a wave traveling inside the microsamples is in phase with itself after having been internally reflected at the interface with the surrounding medium. The analysis of such resonances, which occur for certain values of the size parameter X= ITTYIX, where r is the radius of the microsample, is very well documented (Owen et al., 1982). Calculations show that these resonances should lead to very sharp peaks in the Raman and fluorescence spectra of |xm-sized samples (dimensions up to several tens of juim) that are not predicted in bulk samples of the same composition. These peaks would result either from resonance-induced enhancement of the Raman scattering efficiency itself and/or enhancement of the fluorescent background when fluorescent species are embedded in the sample. Fortunately, the resonances described above have never been observed in the usual micro-Raman experiments. Their absence is explained both by imperfect particle or heterogeneity geometries (complex structures inhibit phase relations between travelUng waves) and the strong optical coupling into the sample substrate, which is for a heterogeneous sample that part of the

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sample which is not illuminated. Both factors preclude the observation of the morphology-dependent effects. Hence, resonances in the inelastic scattering (Raman and fluorescence) spectra of juim-sized samples have only been observed when special experimental conditions (e.g. levitated liquid droplets, glass and polystyrene perfect spheres supported by smooth substrates, and ends of cylindrical optical fibers) have been employed. In these cases the samples have well-defined geometries with virtually no optical coupling into the substrate, if present (Thurn and Kiefer, 1985). Morphology-dependent effects have not, thus far, impaired the analytical usefulness of micro-Raman spectroscopy. However, at fxm sample dimensions orientation effects are far more important (see Chapter 1, Section VIII). Indeed at that scale most of the microcrystals or crystalline domains have well-defined crystallographic axes. Hence, the relative intensities of the Raman bands (compared with those observed in polycrystalUne bulk samples) depend strongly on the orientation of the samples with respect to the incident polarization, as defined by the electric field direction of the laser beam at the sample and the polarization vector of the scattered fight. If no analyzer is used, the latter parameter is determined by the axis of the entrance slit of the monochromator. Care must be taken when qualitative (and quantitative) comparisons of the spectra of microsamples are made with reference spectra obtained from polycrystalline bulk samples, especially for the Raman bands which correspond to totally symmetric modes of vibration. Finally, for samples which contain crystallites whose dimensions are far below the exciting wavelength (nanophases ranging from several tens to a few hundred nm), band shifts and broadening are expected due to finite crystalUte size (relaxation of the K = 0 selection rules), surface pressure and nonstoichiometry. New bands may also be observed which are due to surface and aggregation effects (Bobovich and Tsenter, 1982; Pigenet and Frevet, 1980).

II. INORGANIC SOLIDS A. Catalysts L

Introduction

Metal oxide yAl203-supported catalysts to which an NiO or CoO promoter is added have been extensively studied because of their important industrial applications. In particular, after activation, they are employed in hydrodesulfurization (HDS) and hydrodenitrogenation (HDN) of petroleum and coal products. The precursor oxides, which are the catalysts before activation by sulfidation, are generally prepared by the pore-filUng method with the use

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of yAl203 extrudates according to the following steps (Payen et al., 1986): (i) Impregnation with ammonium heptamolybdate solution, (ii) Drying at temperatures above 373 K, followed by calcination at 773 K for several hours, and (iii) Promoter impregnation with Ni or Co nitrate solution, drying and final calcination at 773 K. The metal and promoter loadings are given in oxide (i.e. M0O3, NiO, CoO) weight per cent. 2. Characterization by Vibrational Spectroscopy In addition to surface characterization techniques (see Chapter 5) such as X-ray photoelectron spectroscopy (XPS) and ion scattering spectroscopy (ISS), vibrational spectroscopy has proven to be a very useful method for characterizing the supported phases themselves. As these catalysts are not transparent below 1000 cm"^ due to absorption by the 7AI2O3 support, Raman spectroscopy is preferred to infrared spectroscopy for obtaining data. Conventional laser Raman spectroscopy has been used to analyze oxide-supported catalysts, but a large number of the results reported in the literature have been obtained with the use of micro-Raman spectroscopy. The advantages of a Raman microprobe for the study of 7Al203-supported catalysts are as follows: (i) In the case of absorbing samples the efficiency of the excitation and collection of hght is far higher than in the conventional instrument (see Chapter 3), (ii) The confocal configuration used is very efficient in reducing background emission, and (iii) The micro-Raman instrument offers the possibihty of employing controlled temperature and controlled atmosphere cells. Such cells for optical microscopy are currently available or can be made in the laboratory. They facihtate the study of sohds in situ under a wide variety of atmospheric conditions and at temperatures ranging from that of Uquid nitrogen to 1500 K. 3. Raman Spectroscopic Analysis of Precursor Oxides The understanding of the function of alumina-supported oxomolybdate catalysts in their oxide states owes much to results obtained by the use of Raman spectroscopy. It was shown by Brown et al. as early as 1977 that even

246

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for loadings lower than that required to saturate the first monolayer, good Raman spectra could be obtained. According to Payen et al. (1986), the observed Raman spectra are complex in nature, as they involve mainly surface effects and loading inhomogeneity. A significant amount of work has appeared in the literature and an overall interpretation of the Raman bands associated with supported oxides has emerged (Payen et al., 1987), namely: (i) At loadings of <5.5 wt% M0O3, only monomeric (M) molybdate species are present on alumina supports. Transformation of the heptamolybdate anions occurs during the wetting of the support, (ii) At higher loadings (>14wt% M0O3), heptamolybdate (HM) aggregates are present. Similar results are observed when loading 7AI2O3 with tungsten and vanadium. In situ measurements at temperatures up to 773 K show that the species M and HM are stable; this stability is thought to result from interaction with the support. During thermal treatment the dehydration and rehydration of the supported species can be followed by the shift of the stretching vibration of the Mo-Ot bond, which occurs between 900 and 1090 cm~^. (Ot denotes a terminal oxygen atom which is not Unked to other atoms.) In the VI"^ oxidation state, molybdenum may adopt various configurations between tetrahedral and octahedral. Hence the shift of the Mo-Ot bond stretching vibration which corresponds to the monomer or heptamer species will depend not only on the ligand heterogeneity (relative to the number of bridging oxygens), but also on the coordination heterogeneity (unsaturated Mo^^ due to ligand vacancies). Both the ligand and coordination heterogeneity effects lead to an increase in the Mo-Ot stretching frequency (see Fig. 1). Thus, by recording spectra in the 900-1090 cm~^ range, it is shown that: (i) Calcination around 773 K reinforces the link between the H and HM species and the support through dehydration. The presence of a promoter (Ni or Co) enhances the phenomenon by faciUtating dehydration. (ii) Calcination at higher temperatures leads to the formation of AI2 (M004)3.

(iii) Ageing of the catalysts after calcination is a hydration process which always leads to the supported hydrated species

Movjf °' . OH (iv) This hydration-dehydration scheme seems to be general and has been observed in other yAl203-supported oxides such as WO3 and V2O5.

Application nb 0

nb nb nb 0 0 0 \ 1/

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247

b nb b 0 0 0 \ l /

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to IVIaterials Science

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// 00 1 1

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X X

1

_>

M06 +

//l\\

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Figure 1 Characteristic Raman frequencies of the molybdenum ion in various oxidation states. The subscripts b and nb denote bridging and nonbridging oxygen atoms, respectively.

4. Bronsted Acidity of Supported Oxides Pyridine is a model molecule which is often used to measure the surface acidity of soHds. In the hydrated form, precursor oxides possess a hydroxyl group bonded to the supported metal. Pyridine chemisorption through the formation of the pyridinium ion is a good way to demonstrate the acidic

248

P. Dhamelincourt and S. Nakashima 1014

(c)

b)

(a) N)cm-i

Figure! Pyridine chemisorption on a Ni-W catalyst, (a) Fresh catalyst, (b) after increasing time of contact with pyridine, (c) after desorption of physisorbed pyridine by N2 purging. character of the OH group. With the use of a controlled atmosphere and controlled temperature cell, Raman measurements can be made during the flow of the N2-pyridine mixtures on powder catalyst (Payen et al., 1982). On molybdenum- or tungsten-supported catalysts, the same observations have been made (see Fig. 2). At first, a band at 1014 cm"^ appears which is characteristic of the chemisorbed pyridine (pyridinium ion). Then, on increasing the contact time, bands at 1032 and 990cm~^ appear which are characteristic of physisorbed pyridine. Subsequent purging with pure N2 leads to the desorption of physisorbed pyridine only (the bands at 1032 and 990cm~^ disappear, whereas that at 1014 cm~^ is unchanged). Therefore, the supported polymolybdate or polytungstate species have acidic Bronsted sites if no pretreatment to desorb water has been performed prior to the absorption experiments. 5. Sulfidation of Precursor Oxides The precursor oxides have to be sulfided (activation step) before being used in the catalytic reactor. This activation process yields supported sulfide

Application to Materials Science

249

(a) (cm - i i

15

M-

380

10J

385

yJ

\

J

L.

L 12

8

% M0O3 (b)

L (nm) 4

-L

J-

8 %Mo03

12

Figure 3 (a) Wavenumber and width of the E2g band and (b) average length of an M0S2 crystaUite versus Mo loading.

particles. The same controlled atmosphere and controlled temperature cell can be used to make in situ Raman analyses of the surface phases appearing during the sulfidation of oxides by H2/H2S or N2/H2S mixtures. During sulfidation intermediate sulfides (MS3) and complex oxysulfides [(MoS2(S2)n)^~] have been identified (Payen et at., 1989). After complete sulfidation, nanocrystallites of M0S2 and WS2 are formed whose dimensions correlate well with the oxide loading for hydrated catalysts (see Fig. 3).

250

P. Dhamelincourt

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6. Conclusion Raman spectroscopy is a sensitive method of monitoring the state of 7Al203-supported catalysts before (precursor oxides) and after activation by sulphidation.

B. Ceramics

1.

Introduction

The Raman microprobe is particularly well adapted to the analysis of ceramics, as it allows the investigation of highly localized volumes in ceramic microstructures. The dimensions of these volumes are comparable to typical grain sizes. This situation is in contrast to that of conventional X-ray diffraction techniques, where the probed volume cannot be localized. Moreover, microphases and inclusions can usually be observed directly under the microscope with the use of conventional illumination techniques (because of differences in reflectivity or color). These elements are normally not visualized by scanning electron microscopy. 2. Polyphase Ceramics (a) Silicon and boron nitride Two polymorphs of silicon nitride (Si3N4) are known to exist {a and 0). The powdered a form is usually the starting product employed in the manufacture of hot-pressed or injection-moulded silicon nitride alloys. The j8 structure is formed during hot pressing. Thus the a-to-j8 phase transition is often used as a monitor of the extent of the hot pressing process. As the spectra of the two structures are completely different (a-Si3N4 is characterized by bands at 262, 365, 514, 670 and 850 cm~^; j8-Si3N4 exhibits a triplet with components at 185, 210 and 230 cm~^), they provide a convenient means of distinguishing the structures. In the same way silicon oxynitride (Si2N20), which often coexists with the nitrides, is easily identified by its Raman spectrum (bands at 187 and 254cm~i). The ultra-hard borazon ceramic is the cubic phase of boron nitride (j8-BN or Z-BN) produced at high pressure and temperature. This material is characterized by the TO and LO modes of the cubic lattice which appear at 1055 and 1306 c m ~ \ respectively. Conversion of the cubic phase to the hexagonal one can be easily seen because the latter, which is isoelectric with graphite, has only a single band at 1367 cm~^.

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(b) Partially stabilized zirconia (PSZ) Zirconia (Zr02) is normally monoclinic at room temperature and transforms to the tetragonal and cubic forms at higher temperatures. However, these higher-temperature structures can be stabiUzed by mixing the starting material with additives such as alumina, magnesia, ceria or yttria. Multiphase ceramics based on the constrained, metastable, tetragonal zirconia constitute a class of technically important materials due to their mechanical properties (flexural strength, toughness, wear resistance, etc.). In recent years considerable effort has been employed in the research and development of these ceramic materials for use as engine components. The toughening mechanism in PSZ depends on a volume expansion and sheer strain that occur when tetragonal zirconia transforms irreversibly into the monocHnic form. This transformation is initiated by the stress fields that form ahead of any crack propagating in the material. The size of the transformed zone is an important parameter used to model the enhanced toughness derived from the transformation. Thus, the determination of the size of the transformation zone is of prime importance and imphes that the relative concentration of the monoclinic phase should be obtained with a good spatial resolution. The monochnic and tetragonal polymorphs of zirconia have distinct and characteristic Raman spectra. In particular, over the range 100-300 cm"^ the monoclinic doublet at 181 and 192 cm~^ is well separated from the tetragonal bands appearing at 148 and 264 cm""^. This result permits the monoclinic concentration to be measured with the use of a relation proposed by Clarke and Adar (1982), namely tl81_j_2l92 ^m

77/0^148 , Ci264\

, ^ 1 8 1 , ^^192

V-^^'

where 3 ^ and 3^ are the integrated intensities of the characteristic bands of each phase (refer to superscripts) and F is a correction factor to allow for the increased Raman cross-section of the monochnic phase with respect to the tetragonal one. Most of the experiments reported in the hterature use the Vickers hardness tester to produce cracks in the ceramic material. The focused laser beam is first positioned on the indentation cracks and then at successively greater distances from the crack. At each point, a Raman spectrum is recorded and the monochnic concentration is evaluated. Plots of the relative monochnic concentration as a function of distance from the stress-induced crack can be estabhshed for different indentations and materials. With the use of a Raman imaging technique, Veirs et al. (1990) obtained maps which give the monoclinic fraction in phase-transformed zones surrounding cracks induced in magnesia-stabihzed zirconia.

252

P. Dhamelincourt

3. High-Tc

and S. Nakashima

Ceramics

(a) Introduction Since the high critical temperature superconductors were discovered in 1986, there has been intense interest in their investigation and characterization by Raman spectroscopy. Many superconducting oxides are now known with various critical temperatures. However, the most interesting ones are those with Tc above the liquid N2 temperature (Tc > 77 K) because of their practical applications (see Fig. 4). Their technology is the same between 77 K and room temperature. (b) Structure of high-T^ superconductors and Raman spectroscopy The common feature of all high-r^ superconductors is that these compounds are copper-oxide-based ceramics for which CUO2 planes are present in a more-or-less oxygen-deficient perovskite structure (see Fig. 4). In these CUO2 planes the electrons which are missing from the closed oxygen shell are responsible for the superconductivity. Thus, most of the Raman work on high-Tc materials has been devoted to phonon characterization because of its possible application to the investigation of the mechanism of superconductivity and, more particularly, to the study of vibrations in the *superconducting' CUO2 planes. [For a review in the field see Ferraro and Maroni (1990).]

T{K) 130 A2M2Ca„_^Cu„02„44 n = 1-3 A = BiorTI M = Sr or Ba

120 110

r

oj

100 O

YBa2Cu30y

90 80 LIQUID

N2 BARRIER

70 k

(a) Figure 4 Superconducting oxides with T^ above liquid nitrogen temperature, (a) Composition and (b) structure.

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253

In addition to the fundamental studies of phonon and related electronic properties, Raman spectroscopy has been extensively used to evaluate the quality of variously prepared high-Tc materials (e.g. bulk powders, thin films and single crystals). (c) Interest in Raman microspectroscopy The potential appUcations of high-r^ superconducting materials are mainly in the field of superconducting microelectronics [superconducting, quantuminterference devices (SQUIDs), superconducting microwave and sub-mm devices, etc.]. But this possibility impUes the fabrication of very-high-quality superconducting thin films. Micro-Raman spectroscopy cannot be used directly as a test of superconductivity, as unequivocal connections between specific vibrational modes and superconductivity have not been made. However, this technique is particularly well adapted for controUing the microchemical structure of the films (compositional heterogeneity and impurity phases), as well as the quality of the epitaxy. The MBa2Cu30(7_;^.) compounds, which have been the most investigated, provide a good illustration of the power of micro-Raman spectroscopy as a controlling technique. These compounds are synthesized with the use of the ternary system BaO(BaC03)-M203-CuO, where M may be any of the rare-earth metals (Y, Gd, Ln, etc.). The yttrium compounds are the best-known members of the series, where Yi 2,3 is the common name given to the yttrium-based compounds (YiBa2Cu3). When x is close to zero, these compounds are superconducting at Tc = 90 K and have an orthorhombic structure. On the other hand, when x is close to unity they are semiconductors with a tetragonal structure. There is now a general agreement that the wave numbers of the five Raman-active Ag modes of the orthorhombic phase are: 502 cm-^' 436 cm-^^ 335 cm" ^ 146 cm-^^ 115 cm-^^

0(IV) 0(II)-Cu(2)-0(III) 0(II)-Cu(2)-0(III) Cu(2) Ba

(axial motion) (in-phase bending motion) (out-of-phase bending motion) (axial motion) (axial motion)

In the early stages of this work many studies were made on poorly controlled materials. Thus, the assignments of phonon symmetries were often ambiguous, or even erroneous, due to the presence of impurities (see below). (d) Characterization of impurity phases Impurity phases are byproducts of the processes which lead to the preparation of superconducting MB CO materials. Micro-Raman spectroscopy may

254

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Table 1 Observed Raman wavenumbers of MB CO materials. Phase

Color

Wavenumbers (cm~^)

Y2CU205

Blue-green Green Black Black

210, 315, 390, 480, 605 265, 330, 395, 516 640 640, 585

YzBaCuOj BaCu02 BaCuO(2+J

be used to characterize impurity phases inside the targets used in sputtering techniques (e.g. DC or RF diode, DC magnetron, or laser), as well as inside the superconducting thin film itself. Though many of the impurity phases have a number of Raman Hnes close to, or coincident with those of MBCO, they are now well characterized (Etz et aL, 1991), as shown in Table 1. (e) Stoichiometry

monitoring

The success of sputtering techniques is a result of their ability to produce homogeneous, stoichiometric thin films on substrates. Post-deposition annealing is generally made in order to optimize the superconducting properties of these films. The critical temperature T^ is very sensitive to the oxygen stoichiometry. Furthermore, thermal annealing under atmospheric-oxygen pressure ensures the reoxygenation of the oxygen-deficient sputtered films. Some modes of vibration of the sample are very sensitive to the oxygen content. In particular, the mode at 500 cm~^ can be used to monitor the homogeneity of the stoichiometry of the films. A number of Raman studies have shown that the frequency shift of this mode is well correlated with the oxygen content (Burns, 1991; Huong, 1991). This observation has been used to test in situ the homogeneity of the film on the jjim scale. (f) Epitaxial quality Oriented superconducting thin films present a very selective anisotropy in their polarized Raman spectra. In particular, spectra recorded with incident and scattered polarization along the c axis {zz spectrum) are quite different from those with polarization along the a ov b axes {xx or yy spectra). With the aid of polarization measurements, it is possible to determine the orientation of any surface and thus to establish the orientation of the film on the substrate. (g) Conclusion Although micro-Raman spectroscopy does not provide a direct test for superconductivity, it is an excellent tool for characterizing the quality of thin superconducting films deposited on substrates.

Application to Materials Science 255 C. Protective Coatings

1. Polycrystalline Diamond Coatings The fabrication of diamond films by chemical vapor deposition (CVD) and, more recently, by plasma-enhanced, chemical vapor deposition (PECVD) at low pressure, has opened potential applications in numerous hightechnology areas. A considerable effort has been made in the perfection of these techniques (Bachmann et al., 1991). Diamond films are produced in order to take advantage of the well-known properties of this substance, which include high thermal conductivity, hardness, chemical inertness and electrical resistance. However, in optical applications it is their transparency that is important, not only from the UV to the far IR, but also in the X-ray region. Micro-Raman spectroscopy provides several key advantages for the investigation of carbon films deposited with the use of any of the CVD techniques. In addition to its spatial resolution, which permits the study of individual microcrystals as well as thin films, Raman spectroscopy can distinguish the various forms of carbon. Thus, carbon with sp^-type bonding (diamond), carbon with sp^-type bonding (graphite and carbonaceous materials) and carbon in mixtures of these two types of bonding (diamondlike carbon) can be characterized by their Raman spectra (Sarvides, 1986). Films prepared by vapor deposition (evaporated or sputtered carbon) are typically diamond-Uke, amorphous carbon films (DLC). On the other hand, films prepared by PECVD methods (DC, RF or microwave plasmas) are either crystalline diamond or DLC films, depending on the conditions of deposition, i.e. nature of the plasma, nature and temperature of the substrate, flow rate and current density (Piano and Adar, 1987). Diamond and perfect graphite are each characterized by a single Raman line which appears at 1332 and 1580 cm "^, respectively. However, when the graphite lattice is disordered, a second line appears at 1360 cm~^ which grows in intensity with increasing disorder (Beny-Bassez and Rouzaud, 1985). Furthermore, both bands broaden as the disorder increases (see Fig. 5). The Raman spectra of DLC differ notably from those of graphite and amorphous carbon (Sarvides, 1986). The DLC spectra are characterized by a very broad band centered at 1530 cm~^, with a more-or-less distinct shoulder at about 1400 cm" 1 (see Fig. 5). An important feature of the Raman spectrum is that it is very sensitive to carbon materials having sp^-type bonding. The Raman cross-section of these materials is far higher than that of diamond, thus small amounts of graphite or diamond-Uke carbon mixed with diamond are easily detected. For example, films that appear to be purely polycrystalline diamond on the basis of electron diffraction results often exhibit bands that correspond to disordered carbon (graphitic or DLC).

256

P. Dhamelincourt and S. Nakashima

1500 Wavenumber

Figures Characteristic Raman spectra of carbon materials, (a) Pyrolitic carbon (highly oriented graphite), (b) polycrystalline graphite, (c) amorphous carbon and (d) diamond-like carbon.

2000

1500 Wavenumber

1000

500

(cm-l)

Figure 6 Raman spectra of a diamond coating on an Si substrate, (a) Single microcrystal, (b) grain boundary. An example of the analysis of a polycrystalline diamond film deposited on an Si substrate is shown in Fig. 6. The spectrum recorded from one microcrystal (Fig. 6a) exhibits the well-characterized sharp diamond line at 1332 c m ~ \ together with very weak bands which are characteristic of graphitic carbon. On the other hand, the spectrum recorded from an area where microcrystals are not adjacent (Fig. 6b) is characteristic of DLC mixed

Application

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257

with diamond. It is worth noting that the spectum of DLC always exhibits an increase in the background due to luminescence emission induced by diamond-lattice imperfections (Etz et al., 1988). Micro-Raman spectroscopy is thus a rapid and sensitive method of characterizing the quality of diamond films and other carbon coatings. The Raman microprobe technique was apphed to the characterization of diamond films by Bonot (1990), who measured the Raman spectra of individual crystaUites of various shapes. For diamond crystallites which are well faceted, the spectra show only the Raman component, but the bands are broader than those obtained from natural diamond. Ager et al. (1991) have studied the frequency and shape of Raman bands for a number of crystallites in diamond films grown by chemical vapor deposition. With the use of a two-dimensional detector they obtained 500 data points from different positions on each of the single films grown under different conditions. It was found that the Raman frequencies and bandwidths are correlated and that the films with higher frequencies have larger bandwidths. 2. Silica Coatings EthylsiHcate paints, charged or not with zinc particles, provide excellent protection against corrosion of steel structures attacked by water or chemicals. The sol-gel transformation of ethylsilicate leads to amorphous sihca, a very inert material. However, it can be apphed only to steel which has been previously sand-blasted in order to permit a mechanical linkage between the silica coating the steel surface. Recently, a new process has been developed (Dhamehncourt et al., 1989; Mayot et al., 1989) which permits both unpolished and polished steel to be coated with ethylsilicate paints. After dipping thlesteel structure in a bath of phosphoric acid, an ethylsilicate prehydrolyzate is vaporized at ambient temperature, resulting in bonding of an amorphous-silica coating to the metal. The chemical phosphatation pretreatment insures that the sol-gel transformation starts from the metal surface. By introducing the correct water vapor pressure in the medium surrounding the film, the reactions are carefully controlled to ensure that the film becomes dense without bursting as residual solvents are released during the densification. The coatings obtained in this manner (with thicknesses varying from 10 to 100 |xm according to the conditions of deposition) offer exceptional electrical insulation and thermal shock strength. Micro-Raman spectroscopy can be used to monitor the extent of silica formation and to characterize the nature of the compound formed at the substrate-coating interface (Mayot et al., 1990). Ethyl residues are well characterized by sharp bands appearing between 3000 and 1000 cm" ^, whereas amorphous silica exhibits wide bands near 500 and 1100 cm~^.

258

P. Dhamelincourt and S. Nakashima

3500

3000

_L

_L

2500

2000

Wavenumber

1500

1000

500

( cm-i)

Figure 7 Micro-Raman study of the interface between sheet steel and siHca coatings showing the strong condensation of ethyl polysilicate induced by the phosphatation pretreatment. Figure 7 presents the results of an analysis of a section of polished sheet-steel a few hours after the ethylsilicate paint deposition. It shows that at the surface the dissolution of the iron phosphate [Vivianite, Fe3(P04)2. 8H2O] has induced a strong densification of ethylsihcate in the first few ixm above the surface. In this region the spectra of the ethyl residues are barely observable. The process creates an interphase between the metal and the silica network which is responsible for the adhesion of the coating. Near the surface the densification process is not achieved. This result is clearly evidenced by the presence of the strong Raman bands of the ethyl residues.

III. MICROELECTRONICS AND SEMICONDUCTORS A. Introduction The Raman microprobe provides a powerful technique for the investigation of semiconductor materials and the analysis of problems in microelectronic devices. This method is a nondestructive one which is important for the characterization of semiconductors with composite structures, ceramics consisting of grains, heterogeneous and device structures, etc. A Raman microprobe measurement is not limited to the study of a local

Application to Materials Science 259

point in bulk materials and small particles. Recent developments in Raman technology have enabled one- or two-dimensional images to be obtained (see Chapter 4). Raman imaging provides information on the spatial distribution of physical quantities in materials such as strain, atomic fraction in mixed crystals, impurity concentrations, free carrier concentrations and local crystallographic orientation. This information is useful not only to evaluate the quahty of a sample, but also to infer the relevant dynamical processes, e.g. growth of crystallites, atomic diffusion and reactions at interfaces or surfaces. In an earher report (Nakashima and Hangyo, 1989) some results were presented on semiconductor characterization with the use of Raman microscopes. This section describes some further developments in this area, focusing attention on the Raman imaging technique. B. Raman Microprobe Measurements

Some precautions are necessary in the application of Raman microprobe measurements. Therefore, several of the problems which are relevant to micro-Raman studies will be briefly described in the following paragraphs. 1. Heating Effects The temperature rise in materials due to laser illumination under a microscope presents a serious problem for the evaluation of strain from observed Raman frequency shifts. The temperature variation not only produces shifts of Raman peaks, but local expansion of the heated region also causes additional strain (Liarokapis and Anastassakis, 1988). The frequency variation of the first-order Raman line of Si is about 0.02 cm~^ per degree at room temperature. This shift corresponds to that of the Si Raman line under a pressure of 0.1 GPa. Accordingly, the determination of the local strain in crystals requires that the Raman microprobe measurements be carried out at minimum laser powers. A point-illumination method is widely used for Raman microprobe measurements of semiconductors because high spatial resolution can be obtained. However, this method results in heating, and possible sample degradation, even when low laser power levels are used. Particular care should be exercised in the measurement of powders and thin films on insulators, because their thermal diffusion is poor. As the laser power level is decreased, Raman peaks shift in general toward lower frequencies. Optimum laser power can be determined if a level can be found below which the Raman peak does not shift. Huang et al. (1990) observed the Raman spectrum of Si with the use of a power of 0.05 mW fxm~^ in order to avoid the heating effect.

260

P. Dhamelincourt and S. Nakashima

2. Oblique Incidence When a wide-aperture objective lens is used a large fraction of the laser beam enters the sample at large angles with respect to the surface normal; this oblique incidence results in an apparent breakdown of the Raman polarization selection rules (Turrell, 1984; Mizoguchi and Nakashima, 1989). This situation is the same for the scattered hght (see Chapter 2). Therefore, these effects should be taken into account in the determination of crystallographic orientation of crystals by Raman microprobe polarization measurements. The use of an objective lens with a small numerical aperture or the rejection of the light at large oblique incidence with the use of a suitable diaphragm is desirable for polarization measurements. 3. Depth Profiling The depth resolution for semiconductors which are opaque to laser Ught is limited by the optical penetration depth. In order to obtain Raman spectra with high depth resolution, the following methods have been used: (i) One observes Raman spectra with the use of various exciting Unes which have different penetration depths. The resulting bandshape change is analyzed with the aid of a model based on the convolution of the penetrating depth of the light and the depth dependence of the Raman bandshape (Shen and Pollak, 1984; Hang et al., 1987). (ii) With the use of beveled samples, Raman spectra of the beveled edge are observed as a function of position by translating the sample under the microscope. The spatial variation of the spectrum is analyzed by the convolution method similar to method (i). Depth profihng of a beveled specimen has been studied with strain and disorder in the GaAs epitaxial layer on Si (Huang et al., 1987), strain in Ge;».Sii_;»;/Si-strained superlattices (Chang et al., 1988), strain in laser-annealed amorphous Si (Inoue et al., 1986), interface disorder of a GaAs/Si heterostructure (Mlayah et al., 1990) and composition in Al;^.Gai_;^. as mixed-crystal layers (Abstreiter et al., 1978). (iii) Depth profiles are obtained from Raman measurements by the use of successively etched surfaces (Kakimoto and Katoda, 1982; Holtz et al., 1988; Roughani et al., 1989). C. Ion Implantation and Annealing Ion beams are widely used in the fabrication of semiconductor devices. Ion implantation is an important technology for impurity doping. The damage to crystals resulting from ion implantation, as well as the recovery of crystallinity after annealing, has been studied by Raman spectroscopy.

Application

1

"1

r

r

to IVIaterials ~i

Science

261

r

CrystdUine Si Implanted with As* 2x10^0111^2

1.5X10 Cm^,.. ^..^u;,;;^,..:^^.,^.

..^..A. 7U \ < ^ O

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9x10 cm

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x2.5 ^'•*V^<_...,s vC/)

x1

Pure Si J AOO

I A20

I A40

^60

WAVE

'^80

500

520

SAO

NUMBER ( c m - ' )

Figure 8 Raman spectra of As'^-implanted Si for various dosages. The implantation of energetic ions produces disorder in crystals and eventually converts the crystalline state into an amorphous one. The Raman intensity of the crystaUine component decreases v^ith increasing damage. With increasing dosage levels of implantation, the crystaUine component

262

P. Dhamelincourt

and S. Nakashima

disappears completely and a broad amorphous band is observed, as shown in Fig. 8. This observation enables the dosage to be evaluated from Raman intensity measurements. Recently, focused ion beams (FIB) have been used to carry out highresolution Hthography and impurity doping in selected areas without the usual photomask and resist steps. The Raman microprobe technique has been applied to the characterization of local regions damaged by FIB implantation. Silicon crystals implanted with a 200 keV FIB of diameter 0.1-0.2 jxm were prepared by Mizoguchi et al. (1987). Using a scanning microscope, they measured successively the integrated intensity of the 520 cm"^ component by translating the sample in such a way that the incident laser beam crossed the implanted and unimplanted regions (Fig. 9a). The Raman intensity was found to vary sharply at the boundary between the implanted and unimplanted regions. From this measurement the normalized intensity, which is defined as 3 ^ = (3cryst ~ 3imp)/3cryst? was obtained (Morhange et al., 1974); 3cryst and Simp are the intensities of the unimplanted and implanted regions, respectively. The normalized intensity for Au^"*", Si^"*" and Be^"^ implanted samples is plotted against the dosage in Fig. 9b. It increases with increasing dosage and saturates ( 3 ^ = 1) above a certain level at which the implanted region is completely amorphous. The onset of the rise of the normahzed intensity depends on the mass of the implanted ion for a constant acceleration energy. These results indicate that dosage levels and the degree of damage can be quantitatively determined once the relation between the normalized intensity and the dosage is experimentally obtained. The damage to the surface layer is not uniform and varies with depth. The depth profile of the damage is inferred from the Raman measurements with the use of laser light of different wavelengths. The penetration depth of visible light in Si ranges from 0.5 to 1 fxm; it is comparable to the projected range of the conventional ion implantation in silicon. The normalized intensity ( 3 N ) as a function of dosage is measured for different wavelengths of the laser light. The results are shown in Fig. 10. For the Au^"^ implantation, 3^f does not vary with wavelength, but does change for the Si^"^ and Be^^ implantations. The results of Fig. 10 lead to the conclusions that: (i) The region damaged by the Au^"^ implantation is very close to the surface and the projected range is smaller than the penetration depth of the laser light in undamaged crystals. (ii) For the Si^"^ and Be^^ implantations, the undamaged or partially damaged regions remain at the surface and the projected range is comparable to the penetration depth. The crystallinity of the implanted layers is recovered by post-annealing with laser or flashlamp irradiation and heat treatment in furnaces. One-dimensional images of the Raman spectrum have been

Application to Materials Science 263

W

\ ^ ^

tto 2 LU »2

"^^^

\ \

\

Damaged region \

-A^Av

2

< < Q:

POSITION

1.0

FIB 200 keV -0.5

A:AU2+

I

o : Si2+ n : Be2+

10

10'^

10'^

10'"

10,16

1017

D O S E (ions cm"^) Figure 9 (a) Structure of a silicon crystal implanted with focused ion beams, (b) Normalized intensity as a function of dosage for Au^^, Si^^ and Be^"^ with the use of 200 keV ions (Mizoguchi et al., 1987).

obtained for Si crystals which were amorphized by P"^ implantation and subsequently annealed by flashlamp irradiation for 10 s at 900°C (Mizoguchi et al., 1995). The Raman images are obtained with the use of the Hne-illumination method (see Chapter 4) and a CCD camera as detector.

264

P. Dhamelincourt and S. Nakashima (a)

.fii—^aS^-

z

-a

n

51A5A

O

A880A

A

A579A

Ion dose (ions cnn ) (b)

i

1

1

1

1

« 1

.

1

D

51A5A

O

A880A

A

A579A 1

1

1

.1 . . j

10'*

Ion dose (ions cnn" —I

(c)

.i

T

0.5'

I

Dose (ions cnn )

Figure 10 The normalized intensity of the siHcon band in implanted regions measured at different wavelengths: 4579, 4880 and 5145 A. (a) Au^"^ ion implantation (FIB), (b) Si^"^ ion implantation (FIB) and (c) Be^"^ (Mizoguchi et al., 1987).

Application

to Materials Science

265

(a) 25.0

c-Si band

20.0

3

B

15.0 H

a o .*^ 10.0-1

cluster band

O

C

AH

C

•

5.0

0.0

450.0

500.0

550.0

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(b) / ;c-Si band A-A' Jj*! V cluster band

P^ ^'

c-c -

!•

480

^

• • ^ > ^ -SH^

500 520 Raman Shift (cm')

540

Figure 11 (a) One-dimensional Raman micrograph of the sample annealed at 900°C. (The depth indicates the Raman intensity, black corresponding to high intensity.) (b) Raman spectra at typical areas inside and outside the cluster and near the cluster edge. Illumination time: 10 s; intensity measured at 4880 A (Mizoguchi et aL, 1995). The crystallinity is recovered in almost all areas when the specimens are annealed at temperatures higher than 800°C. However, in sparse regions of a few U | Lm in size, a broad band shifted to the low-frequency side of the crystalline band (520 cm~^) is observed (see Fig. 11). These regions correspond to clusters with a high density of defects. The existence of the defect clusters was confirmed by TEM measurements. The downshift of the Raman band of a cluster may be due to the local stress produced by the high density of defects. The broadening of the cluster band could be caused by the decrease in the phonon lifetimes (lifetime broadening), as well as the nonuniform distribution of the stress field (inhomogeneous broadening).

266

P. Dhamelincourt

and S. Nakashima

(a) JKK

*"-%

Scan 50 cm s"^ 1 O(K/) • Kyz)

c n n

1 K//

>

hC/)

2

LU 1-

2 Z

/ ^^ • • S

r Yd ft bl

If ^J

< 9 f s < w^ Q: - . 1

\i \^ i\\bo

\ \ iV Vtxf

V——

1

•

1

1

DISTANCE(5^m/ldiv)

DISTANCE(5^m/1div)

DISTANCE(5^m/1div)

DISTANCE(5^m/ldiv)

Figure 12 Raman intensity profiles of laser-recrystallized silicon-on-silicon structures (SOSI) at two different geometries x(yy)x and x(y2)jc, where x||
The inhomogeneous recrystallization of amorphous silicon by pulsed laser irradiation was studied by Huang et al. (1990). Regrowth processes under laser heating have also been studied in polycrystalline silicon and amorphous sihcon films. Figure 12 shows one-dimensional Raman intensity profiles of laser-recrystallized stripes in a silicon film which was directly deposited on the (100) surface of a siHcon single crystal and then amorphized (Nakashima et al., 1984). The Raman intensity was measured at 1 jjim intervals for two

Application to IVIaterials Science 267

different polarizations, x{yy)x and x{yz)x, where x||(100), y||(011) and zII(Oil). The crystals axes are referred to those of the substrate. For samples annealed at lower power levels Raman signals are observed at both geometries, while at high anneaUng power, e.g. 7W, the Raman spectra are very weak in the central region of the stripe for the x{yz)x geometry but are measurable at the boundary regions. The following quantity can be introduced: 5=l - ^ = l - | p ,

(2)

yy

where p is the depolarization ratio. This quantity becomes zero for an ensemble of small grains with perfectly random orientations, like randomly oriented molecules in a gas or liquid. It is equal to unity for an annealed stripe having the same crystallographic orientation as that of the crystalline substrate, because 3^^ = 0. Accordingly, the value of S can be used as a monitor of the orientational ahgnment. In Fig. 13, the quantity S is plotted against position. At an annealing power of 3 W, S is less than 0.5 and is almost constant over the whole annealed region. A steep rise occurs in the central region of the annealed zone at 4.0 W. The width of the single-crystalline region increases as the annealing power is increased. Figure 14 shows the two-dimensional images of the Raman intensity for laser-recrystallized silicon films (Mizoguchi et aL, 1986). A nearly flat intensity distribution along the scanning direction of the annealing laser is observed for the two different polarization geometries. This result indicates that uniform recrystallization occurs along the scanning direction and that the crystallographic orientation of the annealed region is the same as that of the underlying crystal. Irradiation by a focused laser beam easily produces the local conversion of an amorphous state into a crystaUine state. The compositional analysis of Ge-Si alloy microstructures formed by this laser-writing technique has been performed by Herman and Magnotta (1987). Damage induced by pulsedlaser irradiation has been observed in siHcon by Fauchet et al. (1985).

D. Determination of Crystallographic Orientation

Attempts to grow thin films of crystaUine semiconductors on insulators have been made by various methods. However, it was found that it is not easy to obtain large-area single-crystal films. The formation of small grains or misalignment of the crystal orientation often occurs. The determination of the local orientation of the crystallites is required in order to understand the growth mechanism of the thin films and to evaluate the crystal quaHty.

268

P. Dhamelincourt and S. Nakashima (a) 1.0

S 0.5

S0.5h

••V

•^.?^*

DISTANCE(5^m/1div)

DISTANCE(5^m/ldiv)

lid)

_(c)

-n

1.0

1.0 {

)

•

1

S 0.5

I •

K •

S 0.5

•

• •

•J,•

• I—1

1

1

1

DISTANCE(5|im/ldiv)

r\

....

1

1

1

1

DISTANCE(5^m/ldiv)

1 1 S 1 which i 1 Figure 13 Profiles of the value are calculated from the data of Fig. 12. (a) Power of annealing laser 3W, 50cms~^ scan, (b) 4W, (c) 5W and (d) 7W (Nakashima et aL, 1984).

The determination of crystal axes by Raman measurements is based on the fact that Raman scattering intensity depends on the polarization directions of the incident and scattered light relative to the crystal axes (see Chapter 1). Several methods have been proposed for the determination of crystallographic axis from Raman polarization measurements, e.g. those by Hopkins and Farrow (1986), Nakashima et al. (1986), Mizoguchi and Nakashima (1989), Huasheng et al. (1986) and Huasheng et al. (1989). In what follows, one of the procedures which was applied to the diamond structure is briefly described (Mizoguchi and Nakashima, 1989).

Application

to Materials Science

\ ^ ^

^

•^

X

269

X(KK)X \ .

^

> cut a c ^^

/ z

K ^

^

^

(a)

^/

X

0\^

x(yz)x

>

o c o c K

\^<^^^^^^A\

^

/ z

^

^

(b) Figure 14 Two-dimensional images of the Raman intensity of SOSI obtained from (a) xiyy)! and (b) xiyz)^ geometries. The power and scanning speed of the anneaUng laser are 6W and 12.5 cm s~^, respectively (Mizoguchi et al., 1986).

270

P. Dhamelincourt

and S. Nakashima

The relative intensity of the Raman-scattered Hght is given by [see Chapter 1, Eq. (7)] 35-|ee«esP,

(3)

where the unit vectors Cg and Cs define the directions of the electric fields of the exciting and scattered radiation, respectively. The Raman tensor components for the F2g mode in the diamond structure are given by ^0 0 0\

0 dL ,0 d 0/

/O 0 d\

/O d 0\

a^ = I 0 0 0 I and a^ = \d

d 0/

\0

d 0 0 .

(4)

0 0/

As the backscattering geometry is usually used in Raman microprobe measurements, the wave vectors of the incident and scattered hght are given by Kg = (—sini^coscp, —sini^sin(^, -cos<^)

and

kg = —kg,

(5)

where ^ is the angle between kg and the (001) axis of the crystal and ip is the angle between the (100) axis and the projection of k^ onto the (001) surface. In a laboratory coordinate system let i/^o t>e the angle between the projection of the (001) axis on the X-Y plane and the X axis and i// {ifj') be the angle between Ce (Cs) and the X axis. The relative scattering intensity can then be expressed in the form 3 = A{^, (p, iljQ.^) + 5 ( d , (p, i/fo:iA) cos2(A' + C(i^, (p, (Ao:
(6)

where A, B and C are functions of i^, cp, ipo and ip. The Raman intensity is measured as a function of the angle for a fixed polarization direction of the incident light. In the experiment the angle if/' is varied by rotation of a Glan-Thompson prism polarizer mounted in the path of the scattered light. Taking i^, (p, and i/^o as adjustable parameters, the fitting of Eq. (6) to the experimental intensity versus ifj' curve can be carried out. The crystallographic orientation is determined from the best-fit values of these parameters. The accuracy of the orientational analysis obtained from the measurements on Si single crystals was better than 2°. However, surface roughness may result in an error in the determination of the orientation. This effect was discussed by Kolb et al. (1991) and Mizoguchi and Nakashima (1989). The method described above has been applied to laser-recrystalHzed Si films on insulators (SOI). Two types of sample, as shown in Fig. 15, were prepared. The first is seeded SOI, for which a polycrystalline silicon film is directly attached to an underlying Si crystal at an opening in the Si02 film. The film is melted with a scanning continuous wave Ar"^ laser (Fig. 15a). The oriented crystallite at the seeded region extends along the direction of laser scanning. The second type of sample is unseeded SOI, for which there

Application to Materials Science 271 LASER SCANNING >

(a) SEEDED SOI

-Si02

UNSEEDED SOI

Si02

(b)

Figure 15 Structure of laser-recrystallized silicon-on-insulator (SOI), (a) Seeded SOI and (b) unseeded SOI. is no opening in the Si02 film (Fig. 15b). The Seco-etched samples showed that there are a number of grains with small and large areas. The large grains lie in the central region of the recrystallized stripes and the small grains are in the circumference of the stripes. The average size of the large grains is about 20 X 200 |xm^ and that of the small grains is a few |xm. Crystallographic orientations of the laterally seeded and unseeded SOI are determined by the polarization Raman microprobe technique. As shown in Fig. 16, the seeded SOI exhibits a variation of the crystal axes with distance from the seeded region along the direction of laser scanning. At the seeded region, the orientation of the recrystallized film is the same as that of the substrate. The (001) axis is normal to the surface. Going away from the seeded region, the crystal axis varies gradually and at the point B, which is 2 mm from the seeded region, the (001) axis of the film is inclined by about 45° with respect to the surface normal. Figure 17 shows the orientations of vectors normal to the surface for various small grains in unseeded SOI. It can be seen that the normal vectors gather in a certain region. One of the reasons for this tendency may be related to the interface interaction between the silicon film and Si02 layer. Local crystallographic orientations have also been measured for laser-recrystallized silicon films by Kolb et al. (1991), Hopkins et al. (1984), Nakashima et al. (1983) and Nakashima and Hangyo (1989).

272

P. Dhamelincourt

and S. Nakashima

Figure 16 Variation of the (001) axis of the recrystalhzed film along the scanning direction of the laser. The measurement was made at intervals of 100 |xm.

Figure 17 Distribution of the surface orientations of small grains in unseeded SOL The open circles show the unit vector along the normal to the surface.

Application to Materials Science 273 Yoshikawa et al. (1991) applied the technique of Raman microprobe determination of crystal orientation to diamond films grown on cubic BN. They confirmed by this experiment that a single crystal of diamond grows on the (100) surface of cubic BN.

E. Distribution of Free Carriers The control of conductivity (carrier concentration, mobility) in semiconductors is important in device fabrication. Contactless and nondestructive characterization of the concentration and mobility of free carriers is desirable. Raman spectroscopy is a potential technique for this purpose. However, plasmons formed by the free carriers do not couple with Raman active modes in centrosymmetric semiconductor crystals such as Ge and Si. Plasmons were observed in highly doped Ge, but their intensities were low (Cerdeira et al., 1984). In noncentrosymmetric crystals such as zincblende and wurtzite-type semiconductors, the plasmon and an LO phonon form a hybridized mode, the so-called LO-phonon plasmon-coupled (LOPC) mode. This mode is Raman active and has two branches, L+ and L_. The Raman frequency, intensity and shape of the LOPC mode depend strongly on the plasma frequency o)^ and the damping constant y. Both o)^ and y are related to the carrier concentration n and mobility ^t, respectively, through the relations w^ = ATTn^l{eo,nf)

(7)

y=T-i = e/(m»,

(8)

and

where e^ is the high-frequency dielectric constant and m* is the effective mass of the carriers. The analysis of the Raman band of the LOPC mode, therefore, enables the carrier concentration and mobility in semiconductors to be measured. The optically determined carrier concentrations and mobihties are consistent with the values obtained from Hall effect measurements (Irmer et al., 1983; Yugami et al, 1987). The Raman microprobe technique can be applied to the characterization of the nonuniform distribution of dopants in compound semiconductors, which are introduced by atomic diffusion, heteroepitaxial growth at high temperatures and ion implantation. The method is also useful in the evaluation of electrical activity of dopants introduced by ion implantation and subsequent annealing. The distribution of carrier concentration and carrier mobility in GaP hght-emitting diodes (LED) have been obtained by Nakashima et al. (1988). The LED diode used had a p"^-n-n"^ junction structure, as shown in Fig. 18.

274

P. Dhamelincourt

and S. Nakashima

20

1

1

AO 60 DISTANCE ( p m )

1

1——1

1

r

I

l

l

1A

- (b)

-

j

12

r 10

M

^ 8-

^ 6 S 4 u. 2 n

-

j

\

H \ ^ \

\

1

rp—O-O—O——O——O

1

20

20

1

1

1

1

_ J

C

J

1

80

100

AO 60 80 DISTANCE ( j j m )

100

AO DISTANCE

60 (urn)

Figure 18 (a) The intensity, (b) bandwidth and (c) peak frequency of the plasmonLO phonon-coupled mode plotted as a function of distance from the outer surface of the GaP LED (Nakashima et aL, 1988).

Application

to Materials Science — 1

1

-|

1

(b)

275 \—

-

o

300

'>

1

\

1 ^

e

\ \ / >o-o-o

u

>• =1-200 1— _j

p*

CQ O

I

n

100

1 "•

_

> n 1 20

AO 60 80 OISTANCE(pm)

100

L 20

^

_!_

1 _ _

AO 60 80 DISTANCE ( p m )

1

100

Figure 19 Distributions of (a) carrier concentration and (b) mobility in GaP LED obtained from the analysis of the results in Fig. 18 (Nakashima et at., 1988).

The Raman spectra were measured at various points in a cross-section of the diodes. In a GaP carrier, damping is large (wpT
F. Strain in Materials

Strain occurs often in materials with heterogeneous structures and in devices fabricated by various processes. The strain in crystals can be evaluated from the observed frequencies of Raman bands compared with those of strain-free

276

P. Dhamelincourt

and S. Nakashima

crystals. The strains have been measured by Raman spectroscopy in the following materials: (i) Semiconductor heteroepitaxial layers and strained superlattices, (ii) Heterostructures consisting of elements with different thermal expansion coefficients, such as silicon on insulators (SOI structures), and (iii) Semiconductor surfaces prepared by poHshing, ion implantation, ion etching, etc. The Raman measurement of strain in semiconductors has attracted much attention because the strain affects electrical properties and sometimes induces defects or dislocations. The distribution of strain depth has been measured with the use of the Raman microprobe and bevelled samples. The crystal quality and strain in laser-annealed, amorphous Si films have been examined by Inoue et al. (1986). The Raman spectra of the beveled portion were measured as a function of the position. The variation of the Raman feature with depth was analyzed by the deconvolution of Raman components arising from the crystalline substrate and the annealed layer. The variation of the residual strain and crystal perfection with depth in the annealed layer was estimated by this procedure. The distribution of strain depth has also been measured for GaAs epitaxial films on Si with the use of a bevelled specimen (Huang et al., 1987). Silicon films grown on insulators have residual strains owing to the differences in the thermal expansion coefficients between silicon and the underlying insulators. The strain varies steeply at the edges and interfaces of these composite materials. Zorabedian and Adar (1983) have measured the strain distribution in laser-recrystallized silicon films on silicon oxide. The strain in silicon with a LOCOS (local oxidation of silicon) structure has been evaluated by Kobayashi et al. (1990). The nonuniform distribution of the strain has been measured for many materials: silicon-on-sapphire (SOS) device structures (Breck et al., 1982), patterned SOS (Yamazaki et al., 1984), Ge islands on Si02 (Takai et al., 1984), Ge on Si and Si02 (Fauchet et al., 1987), GaAs on Ge (Fauchet et al., 1987) and a thermally oxidized Si substrate (Miura et al., 1990). The Raman microprobe measurement of trench-isolated silicon islands has been reported by Tomozawa et al. (1991), who obtained the strain distribution in the cross-section of an Si island. The strain distribution in Si substrates with tungsten electrodes has been measured with a sub-|xm spatial resolution by Sakata et al. (1990). They observed Raman spectra as a function of position with the use of a focused laser beam with a 1 |xm spot size. The resulting spectral changes were analyzed assuming that a Raman band is the convolution of the straindependent Raman band and the intensity distribution of the laser beam. The result is shown in Fig. 20. There is a compressive strain in the Si substrate near the edge of the W film. The strain decreases on a sub-|xm scale as the distance from the edge of the W film increases.

Application

to IVIaterials Science

211

300 250 CL

If) 0)

I I 0

0.2

0.4

0.6

0.8

JO 1.0

Distance from the edge, x (//m)

Figure 20 Raman shift of the Si band and the compressive strain versus distance from the edge of a thin W film. The full and open circles indicate the values of the shift of the Raman peak for the two polarization directions (Sakata et al., 1990).

Cheong et al. (1987) have measured residual strains at Si quartz interfaces with the use of a Raman microscope. In order to improve the spatial resolution in the axial direction, an aperture was placed at an intermediate image plane between the Raman microscope and the entrance slit of the monochromator (see Chapter 2, Section VI). It was inferred from the results that the residual strain exists within a small region of the substrate (quartz) near the interface, about twice the thickness of the Si film.

G. Thermal Conversion of SiC Polytypes

Zincblende-structured j8-SiC is converted into a-SiC polytypes by maintaining the samples at high temperatures. This thermal conversion of the polytype does not occur uniformly at the early stages of the process. A nonuniform spatial distribution of the Raman spectra has been observed in thermally converted SiC crystals (Yoo and Matsunami, 1991). Figure 21 shows the Raman spectral profile of j8-SiC after annealing at 2080°C for 3 h . The Raman bands corresponding to the 6H polytype are clearly observed at 789 and 768 cm~^. The Raman band at 789 cm~^ is due to the TO band of j8-SiC, and the TO band of the 6H polytype, which can be observed in this experimental geometry.

278

P. Dhamelincourt and S. Nakashima

760

780 800 WAVENUMBER (cm-^)

Figure 21 One-dimensional spectral profile of an SiC polytype converted at 2080°C (anneal time: 3h).

The Raman intensity ratio of the 768 and 789 cm"^ bands changes from place to place, indicating that the polytype conversion is not uniform. Raman bands corresponding to the 15R polytype bands are found at some locations. These results indicate that the polytype conversion at 2080°C is not spatially uniform. Raman spectral images were also measured for samples annealed at 2200°C for 2h. They show^ed only slight intensity variation Wiih position.

H. Raman Microprobe Measurements of Inorganic Conducting Materials Raman microprobe measurements have been made for SbCls, Br2 and FeCl3 graphite intercalation compounds by McNeil et al. (1985). Depletion of the intercalate was observed within 10 ^Jim of the sample edge in the SbClsgraphite compound. Those authors concluded that the intercalate contracts thermally within graphite as the sample is cooled after the intercalation reaction is completed. Micro-Raman spectra of graphite intercalated with SO3 were obtained by Ladjadj et al. (1985). They observed the Raman spectra as a function of the intercalation time to obtain information on intercalation kinetics. Raman microprobe spectra of graphite fibres with a diameter of 7.6 |xm were measured under uniaxial stresses by Sakata et al. (1988). The polarization-dependent splitting and shift of the graphite Raman peak were observed under tension along the fiber axis.

Application to IVIaterials Science 279 Tungsten silicide films formed on silicon were studied with a Raman microprobe (Codella et al., 1985). The Raman spectra were obtained from siHcide films 8 |xm wide by 20 nm thick.

IV. POLYMERS AND FIBERS A. Introduction For many years the study of industrial polymers with the use of conventional Raman spectrometers was limited by the high fluorescence induced by the visible excitation used. In most cases the weak Raman lines were completely masked by a strong fluorescence spectrum. More recently, FT-Raman instruments which employ exciting lines in the near-IR spectral region have overcome the problem of fluorescence (see Chapter 3). This technique allows the Raman spectra of bulk industrial polymers to be obtained without difficulty. However, when high spatial resolution is required (e.g. analysis of luim-sized defects or of single textile filaments), FT-Raman instruments equipped with a microscope have thus far been limited to a sensitivity which is far lower than that of modern Raman microspectrometers equipped with multichannel detectors and which employ visible excitation. Consequently, FT-Raman microscopes often yield deceiving results. Although micro-Raman spectrometers use visible excitation, when they are well designed the use of a confocal configuration almost eliminates the fluorescence which falls outside of the focal volume. Thus, the Raman spectrum of most industrial polymers can be successfully recorded. B. Identification of Defects The prime consideration of a manufacturer is the continuing satisfactory and trouble-free production of a good-quality product. These criteria are especially important in the manufacture of a textile from the synthetic fibers produced by spinning from the molten polymer. Defects or breaks in the textile filament can perturb the production process and impair the quality and appearance of the product. Typically, the textile yarn is composed of several tens of filaments spun and wound together. For economic reasons the spinning speeds are now on the order of 6000 m per minute (Ogilvie and Addyman, 1980) which implies that the diameter of an individual filament ranges from 5 to 20 \xm. Thus, at this scale the filament breaks caused by particulate contamination may be important when the contaminant becomes a substantial proportion of the material flowing through the spinneret. Filters are mounted before the spinneret which remove particulate matter down to the 5 |uim range. Accordingly, the analytical tools employed must function

280

P. Dhamelincourt and S. Nakashima

in the spatial domain which is less than 5 |xm. A large variety of microanalytical techniques exist which determine the elemental composition of particulates, but molecular characterization is often necessary in order to identify the contaminant material and establish its source. Micro-Raman spectroscopy is particularly well adapted to this kind of analysis because it permits a filament contaminant to be directly identified by focusing the laser beam on it and recording the Raman spectrum. There are two types of inclusion arising from particulate contamination of the feedstock polymer (Ogilvie and Addyman, 1980): (i) Internal contamination from substances which are part of the polymer recipe or produced during processing. For example, titanium dioxide, which is used as an antilustre agent in the textile yarn, and carbonaceous residues produced in the thermal degradation of the polymer, have been identified. (ii) External contamination from various substances acquired during the material-handling processes. Materials identified by micro-Raman spectroscopy include packaging materials (polythene, polyvinyls), insulation materials (fiberglass) and miscellaneous substances (polyamide fibers from workers' clothing, airborne particles such as quartz and calcium carbonate, etc.). C. Analysis of Chemical Composition

The intensity of Raman scattering by a functional group is proportional to its concentration. This relation is invaluable for the determination of the relative concentration of compounds which exist in several isomeric forms. For example, polybutadiene has three isomeric forms (d5-l,4, trans-l,A and syndiotactic-1,2). This material is often used as a blend or copolymer with polystyrene in order to improve the impact strength of the polystyrene. The relative concentration of the three isomers of polybutadene is an important parameter which governs the mechanical properties of the polymer. In the 1600-1700 cm~^ spectral range, the C=C stretching modes of the three isomers appear separately in the Raman spectrum. This fact enables the ratio of these isomers to be determined in situ and microscopic inhomogeneities can thus be detected. Figure 22 shows the analysis of a poly butadienepolystyrene copolymer in which the variation in the concentration of the cis isomer is followed. D. Analysis of Morphology in Polyester Fibers

Polyester textile fibers are made of polyethyleneterephtalate (PET), whose molecular geometry is shown in Fig. 23. In the crystalline state the

Application

to Materials Science

281

1665 (frans)

V (cm ^)

Figure 22 Raman spectra of polybutadiene in the C=C stretching region, (a) At the center of the sample and (b) at the sample boundary.

Trans

Gauche Figure 23 Molecular geometry in PET fibers.

282

P. Dhamelincourt

and S. Nakashima

arrangement of the polymer chain corresponds to a trans conformation of the glycol unit, as well as coplanarity of the carbonyl group with the benzene ring. In the amorphous state, the coplanarity and the trans conformation of the glycol units do not exist; thus, the gauche conformation of the glycol unit is prevalent in the material. However, some trans conformation may still be present. The macroscopic properties of a polyester textile are governed by amorphous and crystalline structural arrangements in the fibers. Thus, in order to improve the mechanical properties of these fibers, they are stretched or drawn during production. This orients the molecules so that the chain axes are preferentially oriented towards the fiber axis. However, the stretching or drawing of the fiber modifies its morphology, as it changes molecular alignment, conformation and crystallization. With the use of polarization measurements and band assignments it is possible to monitor independently molecular alignment and crystallization (Cook and Ogilvie, 1982). MicroRaman spectroscopy allows information on both of these characteristics to be obtained from a single filament. This technique is very important in the detection of drawing faults in fibers which result when they are subjected to the stretching and shrinking processes of the textile industry. Orientation measurements have been well described by Purvis et al. (1973) for PET fibers. For transversely isotropic polymers, such as in a uniaxially drawn PET fiber, a relationship exists between the values of cos^ 6 and cos"*^, where 0 is the angle between the molecular chain axis and the drawing direction. These two functions of 6 can be determined by refractive index measurements (for cos^ 6) and from polarized Raman spectra (for both cos^ 8 and cos^^). In practice, the degree of orientation in polymers is defined by the two averaged Legendre polynomials F2 = 5 ( 3 c o s ^ - l )

(9)

P 4 = f c o s ^ - f c o s ^ + i.

(10)

and

For a completely nonoriented (isotropic) polymer ^2 = 0 (since cos^ 6 = 1/3), while for a perfectly oriented fiber P2= ^ (since cos^^ = 1). The development of orientation can be monitored by studying the 1615 cm~^ band, which corresponds to the C—C stretching vibration of the benzene ring. It is sensitive only to the molecular chain orientation. Polarization measurements on a single filament require it to be positioned with its draw axis Z along the larger dimension of the microscope shde, and the laser beam to propagate along the Y axis. An analyzer is set to transmit the Raman radiation which is polarized perpendicular to the entrance sUt of the instrument and a rotating microscope stage is employed. It is then

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A. Y{ZZ) Y A':Y{ZX)Y

A: Y{XX) Y

Figure 24 Polarization configurations for orientation measurements in polyester fibers. possible to measure three different intensities for ^he 1615 cm ^ band; 3zz? 2zx and 3xx, which correspond to the Y(ZZ)Y, Y(ZX)Y and Y(XX)Y polarization configurations, respectively (see Fig. 24). With the use of the method of Purvis and Bower (1976), Cook and Ogilvie (1982) derived the equations which have to be solved to obtain the orientation functions P2 and P4, namely 3zz = ^(0.169 4- O.512F2 + O.3I8P4),

(11)

2zx = ^(0.093 + O.O66P2 - O.I6OF4)

(12)

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and

3 ^ ^ = ^(0.169 - 0.256F2 + O.II9P4),

(13)

where yl is a constant. When the laser beam illuminates a filament, polarization scrambling due to reflection and refraction can lead to errors in intensity measurements. These errors can be effectively eliminated by immersing the sample in a liquid with a similar refractive index. Ogilvie and Cook (1982) have reported values of P2 obtained from Raman measurements on fibers having different draw ratios, which correlate well with those obtained from birefringence measurements. The degree of crystallinity in PET is obtained by measurement of the bandwidth of the carbonyl-stretching vibration located at about 1730 cm~^. Generally, with increasing crystallinity, the associated conformational bands show decreasing bandwidth. The bandwidth of the carbonyl band in PET is indeed a measure of the conformational disorder due to the rotations of the carbonyl group around the carbonyl phenylene bond (Melveger, 1972). Recent work reported by Adar et al. (1990) shows that with the use of band-fitting procedures the carbonyl band is in fact complex and is composed of two or three bands. The central band at 1726 cm~^ is Unked to crystalHne PET, whereas the other two bands, at 1721 and 1735 c m ~ \ characterize the amorphous phase. The intensity increase of the central band at the expense of the lateral bands explains the profile change and the clear correlation of the width of the multicomponent carbonyl band with the crystallinity of the polymer. Nevertheless, the detailed analysis of this carbonyl band provides more information on the amorphous, oriented polymer (i.e. spin-oriented fiber).

E. Conclusion

Chemical compositions, molecular configurations and conformations in polymers are identified through their vibrational frequencies. Thus, this information can be obtained by FT-IR techniques and, more recently, with the use of FT-Raman accessories proposed by IR instrument manufacturers. However, Raman microscopy remains as a unique, invaluable tool for the analysis of polymers at the microvolume level (a few \LW?). Thus, the nature of defects or inhomogeneities can be readily identified. In the same way, investigation of polymer morphology and quantitative measurements of localized molecular symmetry in oriented polymers are possible from Raman polarization measurements.

Application to Materials Science 285 V. GENERAL CONCLUSIONS Raman spectroscopy was not widely applied to the characterization of materials until the advent of Raman microprobe techniques. With conventional Raman spectroscopy it was not possible to reduce and localize the analysis volume to dimensions commensurate with grain or phase size in microstructures or with the size of the analyzed object itself (e.g. microelectronic devices and fibers). Now the ability to investigate regions as small as 1 |xm in diameter with a tool which yields molecular information enables Raman microspectroscopy to complement existing microanalysis tools (scanning electron microscopy, X-ray microanalysis, etc.) and to be a unique technique for the investigation of organic substances. The examples given in this chapter have been selected in order to illustrate what Raman microspectroscopy can offer in the investigation of both organic and inorganic materials. These examples do not, however, represent an exhaustive hst of applications. Indeed, with today's changing technologies and the rapid appearance of newly engineered materials, the future of Raman microspectroscopy for materials characterization is extremely promising.

REFERENCES Abstreiter, G., Bauser, E., Fischer, A. and Ploog, K. (1978). Appl Phys. 16, 345. Adar, F., Armellino, D. and Noether, H. (1990). Proc. SPIE 1336, 182-193. Ager III, J. W., Veirs, D. K. and Rosenblatt, G. M. (1991). Phys. Rev, 43B, 6491. Bachmann, P. K., Leers, D. and Lydtin, H. (1991). Diamond and Related Materials 1, 1. Beny-Basez, C. and Rouzand, J. N. (1985). Scanning Electron Microscopy 1, 119. Bobovich, Y. S. and Tsenter, M. Y. (1982). Opt. Spectrosc. (USSR) 53, 332. Bonot, A. M. (1990). Phys. Rev. 41B, 6040. Breck, S. R. J., Tsaur, B. Y., Fan, J. C. C , Murphy, D. V. R. F. and Silversmith, D. J. (1982). Appl. Phys. Lett. 40, 895. Brown, F. R., Makowsky, L. E. and Rhee, K. M. (1977). /. Catal 50, 6642. Burns, G. (1991). Solid State Comm. 11, 367. Ceirdera, F., Mestres, N. and Cardona, M. (1984). Phys. Rev. 29B, 3737. Chang, S. J., Kallel, M. A., Wang, K. L., Bowman Jr, R. C. and Chow, P. (1988). /. Appl. Phys. 64, 3634. Cheong, Y. M., Marcus, H. L. and Adar, F. (1987). /. Mater. Res. 2, 902. Clarke, D. R. and Adar, F. (1982). /. Am. Ceram. Soc. 65, 284. Codella, P. J., Adar, F. and Liu, Y. S. (1985). Appl. Phys. Lett. 46, 1076. Cook, B. W. and Ogilvie, G. D. (1982). Microbeam Analysis, 294. Dhamelincourt, P., Ecuyer, M., Lalart, D., Lemaguer, D. and Mayot, A. (1989). French patent no. 8908 510. Etz, E. S., Farabaugh, E. N., Feldman, A. and Robins, L. H. (1988). Diamond Optics, SPIE 969, 86.

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Nakashima, S. and Hangyo, M. (1989). lEE J. Quantum Electron, 25, 965. Nakashima, S., Inoue, Y., Miyauchi, M., Mitsuishi, A., Nishimura, T., Fukumoto, T. and Akasaka, Y. (1983). / . Appl. Phys. 54, 2611. Nakashima, S., Inoue, Y. and Mitsuishi, A. (1984). / . Appl Phys. 56, 2989. Nakashima, S., Mizoguchi, K., Inoue, Y., Miyauchi, M., Mitsuishi, A., Nishimura, T. and Akasaka, Y. (1986). Jpn. J. Appl Phys. IS, L22. Nakashima, S., Yugami, H., Fujii, A., Hangyo, M. and Yamanaka, H. (1988). / . Appl Phys. 64, 3067. Ogilvie, G. D. and Addyman, L. (1980). UActualite chimique. p. 51. Owen, J. F., Barber, P. W. and Chang, R. K. (1982). In: K. F. J. Heinrich (ed.), Microbeam Analysis. San Francisco Press, San Francisco, p. 255. Payen, E., DhameUncourt, M . - C , Dhamelincourt, P., Grimblot, J. and Bonnelle, J.-P. (1982). Appl. Spectrosc. 36, 30. Payen, E., Kasztelan, S., Grimblot, J. and Bonnelle, J.-P. (1986). / . Raman Spectrosc. 17, 233. Payen, E., Grimblot, J. and Kasztelan, S. (1987). / . Phys. Chem. 91, 6642. Payen, E., Kasztelan, S., Houssenbay, S., Szymanski, R. and Grimblot, J. (1989). / . Phys. Chem. 93, 6501. Piano, L. S. and Adar, F. (1987). Proc. SPIE Conf. 822, 52. Pigenet, C. and Frevet, F. (1980). Phys. Rev. 22B, 2785. Purvis, J., Bower, D. and Ward, I. M. (1973). Polymer 14, 398. Purvis, J. and Bower, J. (1976). / . Polym. Set. 14, 1461. Roughani, B., Kallergi, M., Aubel, J. and Sundaram, S. (1989). / . Appl Phys. 66, 4946. Sakata, H., Dresselhaus, G., Dresselhause, M. S. and Endo, M. (1988). / . Appl Phys. 63, 2769. Sakata, H., Hatsydan, T. and Kawai, S. (1990). Proc. 9th Int. Conf. Experimental Mechanics, Copenhagen, p. 1307. Sarvides, N. (1986). / . Appl Phys. 59, 4133. Shen, H. and Pollak, F. H. (1984). Appl Phys. Lett. 45, 692. Takai, M., Tanigawa, T., Miyauchi, M., Nakashima, S., Gamo, K. and Namba, S. (1984). Jpn. J. Appl Phys. 23, L363. Thurn, R. and Kiefer, W. (1985). Appl Opt. 24, 1515. Tomozawa, M., Vasquez, B. and Ikeda, T. (1991). Extended Abstract of the 1991 International Conference on Solid State Devices and Materials, Yokohama, p. 234. Turrell, G. (1984). / . Raman Spectrosc. 15, 103. Veirs, D. K., Ager, J. W., Loucks, E. T. and Rosenblatt, G. H. (1990). Appl Opt. 25, 4969. Yamazaki, K., Uotani, R. K., Nambu, K., Yamada, M., Yamamoto, K. and Abe, K. (1984). Jpn. J. Appl Phys. 23, L403. Yoo, W. S. and Matsunami, H. (1991). Jpn. J. Appl Phys. 30, 545. Yoshikawa, M., Ishida, H., Ishitani, A., Koizumi, S. and Inuzuka, T. (1991). Appl. Phys. Lett. 58, 1387. Yugami, H., Nakashima, S., Mitsuishi, A., Uemoto, A., Shigeta, M., Furukawa, K. and Nakajima, S. (1987). / . Appl Phys. 61, 354. Zorabedian, P. and Adar, F. (1983). Appl Phys. Lett. 43, 177.

Applications in Earth, Planetary and Environmental Sciences Paul F. McMillan, Jean Dubessy and Russell Hemley

I. INTRODUCTION

As in many fields of natural science, the first step in a mineralogical, petrological or geochemical study is usually the examination, description and identification of an object. In the geological sciences the two classic techniques most often used for these operations are optical microscopy and X-ray diffraction. These techniques are often complemented by scanning or transmission electron microscopy, or by electron or ion microbeam analysis to obtain chemical compositions. However, even when they are used in combination, these techniques cannot, for several reasons, be applied successfully to all problems. For example, the sample of interest may be included in another mineral, or may contain a high concentration of light elements (especially H, Li or B), which are not easily amenable to X-ray or electron beam analysis. In addition, traditional examination techniques often cannot distinguish between different polymorphs of a given structure, or determine partial degrees of order. For these reasons, another microbeam method of investigation, which can yield a molecular fingerprint of the sample, and which can be used for the study of crystalline or amorphous materials, is necessary. These requirements have been met by a new generation of Raman spectrometers, the Raman microprobes. These instruments allow the characteristic vibrational Raman spectrum to be obtained routinely from juim-sized regions of crystaUine or amorphous soHds, even when these structures are in the form of inclusions within a host material. One particular type of 'inclusion' study is that which employs micro-Raman spectroscopy to examine samples contained in a diamond anvil cell. This technique, which allows spectra to be obtained from materials in situ at high pressures (and high

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temperatures), is particularly suitable for studies of geological materials under conditions similar to deep planetary interiors. In addition, microRaman techniques can be easily applied to investigate the molecular vibrations of materials containing light elements, and can be used to distinguish different degrees of structural order. It is worth noting that, with the use of the micro-Raman technique, the vibrational spectrum of a mineral can be obtained from the same spot size that is used for compositional determination via electron beam microanalysis (see Chapter 5).

II. PRACTICAL ASPECTS Apart from samples studied as powders or single crystals (single crystals of only a few ixm in dimension can be studied with the use of micro-Raman techniques), most geological samples are in the form of mounted, thick or thin, singly or doubly polished sections, as used for optical microscopic and electron microprobe analyses. Such preparations are convenient for microRaman investigations, provided that any carbon layer deposited for electron microprobe analysis is removed, as this layer exhibits its own characteristic Raman spectrum. It should be noted that Canada balsam must be avoided as a glue for mounting samples because its strong fluorescence under laser irradiation completely overpowers the Raman signal (Pasteris, 1989). On the other hand, mineral plates of 150-300 fxm thickness, which are typically used for the observation of fluid inclusions, are convenient for their micro-Raman investigation. One major practical consideration in micro-Raman spectroscopy is dictated by the working distance and numerical aperture (N.A.) of the microscope objective used to carry out the experiment (see Chapter 2). A high value of the N.A. (>0.6) is usually necessary for efficient collection of the scattered light, so that a useful spectrum, with adequate signal-to-noise ratio, can be collected in a reasonable time. This condition was especially important for work with scanning Raman spectrometers, although it is now less of a consideration. With the advent of instruments equipped with diode-array detection (where considerably more signal averaging is obtained in an equivalent time) or highly sensitive charge-coupled device (CCD) detectors, as well as Fourier transform instruments, this problem has effectively been eliminated. In any case, it is alleviated with the use of the high-N.A., dry objectives commonly used in optical microscopy, which have working distances of a few mm. These objectives can be used in most studies of powdered or bulk samples at ambient conditions. However, this method is no longer possible in the study of samples included in other minerals at depths greater than 75-100 luum with respect to the host surface, or located inside

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in a high-pressure cell (usually a diamond-anvil cell) or a heating furnace, which necessitate much longer working distances. These appHcations require the use of specially designed, long working distance, dry objectives, with as high a numerical aperture as possible, coupled with efficient detection of the Raman signal. Often the loss of light intensity, both from the incident and the scattered beams, is large when dry objectives are employed, because of the high refractive index of the minerals studied (usually in the range 1.5-1.7). This problem can be particularly critical in the observation of inclusions. Immersion objectives with oil or water as immersion media can be used to resolve this problem. The advantage of water immersion objectives is that water does not give rise to a strong Raman signal, provided that axial spatial filtering is employed. Their major disadvantage is the rapid evaporation of the water. Oil immersion objectives use an alkane as an immersion medium. However, the high intensity of their C—H stretching bands around 2900 cm~^ renders this medium of little use for fluid-inclusion studies if an alkane (usually methane) is being investigated. The use of immersion objectives requires a large, flat surface to retain the immersion Hquid, a condition which is not always fulfilled, especially in gemmological studies. Fluorescence is often a major problem in the application of Raman spectroscopy in the earth sciences. The major difficulty is due to electronic fluorescence, excited by the incident laser light, which is often much more intense than the weak Raman signal. This problem is particularly important in the case of iron-containing minerals, or for fluid inclusions with hydrocarbons present (Wopenka et al., 1990). In some cases, prolonged exposure to the incident laser beam will 'bleach' the fluorescence, and a Raman spectrum can be obtained. This effect is most often observed with organic materials (Pasteris, 1988). In other cases it is possible to use a laser wavelength which does not excite electronic transitions. For example, useful spectra of iron-containing minerals and glasses can be obtained with the use of red or yellow excitation (Griffith, 1969a,b, 1974; Mao et aL, 1987; Sharma and Cooney, 1990, 1992; Wang et aL, 1991a). Because the intensity of the Raman scattering is dependent on the inverse fourth power of the excitation wavelength [see Eq. (7) Chapter 1], the use of red laser light results in a severe reduction in signal-to-noise ratio, thus longer acquisition times are required. However, many of the photomultipUers, diode-array or CCD detectors which are available for Raman spectroscopy are optimized for maximum sensitivity in the red spectral region. One elegant solution to this problem is the use of a Michelson interferometer to obtain a Fourier transform Raman spectrum (Chase, 1987). In this experiment, a near-IR laser (for example, the 1.06 |xm fundamental radiation of an Nd:YAG laser) is used to excite the Raman spectrum of the sample. This excitation has insufficient energy to induce transitions between electronic states in many

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minerals; so fluorescence is avoided. The low signal-to-noise ratio per scan is compensated by the multiplex advantage of the Fourier transform method, thus extremely high-quality spectra of samples have been obtained in very short times (Chase, 1987). Another method of overcoming the fluorescence problem is to carry out time-resolved Raman experiments (Kamogawa et at., 1988; Sharma, 1989). Because the Raman effect is essentially instantaneous, whereas electronic fluorescence usually has a much slower response, it is possible to discriminate between the Raman and the fluorescent signals with the use of a gated detection system. Another interesting method for extracting the Raman signal from the spectra of strongly fluorescent samples is based on the digital analysis of the random noise in the total collected signal (Durham, 1989). Micro-Raman spectroscopy with the diamond-anvil cell can be difficult due to the fluorescence of the diamond windows, which often overpowers the weak Raman signal. The various solutions to this problem include the careful selection of diamonds (type II) for low fluorescence in the spectral range of interest, direction of the incident laser beam at approximately 45° with respect to the collection optics, and careful spatial filtering before the spectrometer entrance (Hemley et at., 1987a). Raman spectroscopy at high temperatures is difficult because of the strong thermal emission of the sample, for which the intensity at a given wavelength increases as the fourth power of the absolute temperature. Once more, long working distance objectives are required, especially for temperatures above a few hundred degrees Celsius, to avoid degradation of the objective (cooUng of objectives can be useful in this case), and spatial filtering is required. In fact, micro-Raman spectroscopy is a technique of choice for high-temperature studies. Because the incident beam is focused on, and collected from, a ixm-sized region of the sample, blackbody radiation from the remainder of the sample and the furnace assembly can be considerably reduced or eUminated by spatial filtering after the microscope. Time-resolved methods can be used to discriminate further between the Raman spectrum, which is excited only on laser irradiation of the sample, and the blackbody radiation background, which is emitted continuously at high temperatures (Sharma, 1989). Finally, studies of isolated small particles, especially ones which are highly colored or are inherently unstable (or metastable) at ambient conditions (e.g. jxm-sized particles of carbon polymorphs, high-pressure phases, minerals containing transition metals), are often complicated because the sample is damaged or destroyed under laser irradiation via absorption of the incident beam. Obvious solutions to this problem are to reduce the laser power or to choose an excitation wavelength which is not absorbed. Another technique which can be useful is to embed the sample in a transparent matrix material such as KBr to ensure good thermal contact with the matrix and thus to dissipate the heat generated.

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III. MINERALOLOGY AND PETROLOGY A. Phase Identification in Natural and Synthetic Samples One of the first steps in the characterization of a natural or synthetic rock sample is the identification of the phases present. This consideration is particularly important in conjunction with geological field studies, as well as in experimental petrology, so that the pressure and temperature at which a given mineral assemblage was formed, and the P-T trajectory which it subsequently underwent, can be established. Such P-T determinations carried out on individual, natural samples are then often used in a second step to reconstruct the P-T conditions on a regional scale, for example, during formation of a mountain belt. In the classic method, all of the mineral and other phases present in a rock sample are identified, along with their textural and spatial relationships. In addition, some assumptions about the equilibrium conditions are usually made. This analysis allows the pressure and temperature of the rock formation to be reconstructed from a prior knowledge of the phase diagram. Much information about the phases present and their relationships can usually be obtained by applying classical optical techniques with the use of the petrographic microscope, although there are limitations to this type of study. Some of these restrictions can be overcome by applications of micro-Raman spectroscopic techniques, especially since the characteristic Raman spectra of different mineral classes are becoming well known and understood (Griffith, 1987; McMillan and Hofmeister, 1988; Sharma, 1989). If an amorphous phase is present, optical microscopy shows only the presence of an isotropic material with a particular refractive index. MicroRaman spectroscopy, on the other hand, can be used to gain detailed information on the structural state of the glass (or gel), its degree of hydration, and can sometimes indicate the presence of sub-microscopic crystals within the glass. This capability can be particularly useful in the study of shocked phases. 7. Identification of Crystalline

Polymorphs

One area in which micro-Raman spectroscopy is an essential complement to optical microscopy is in the identification of crystalline polymorphs, which are difficult to distinguish with the use of optical techniques. The presence of certain polymorphs in a given mineral assemblage can provide important constraints on the P-T conditions encountered by a rock, especially during metamorphism. The Al2Si05 polymorphs (silHmanite, andalusite, kyanite) form, perhaps, the best known examples. These substances can usually be readily recognized and differentiated using optical techniques, but not in all

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Sillimanite

M-^uJjJ Kyanite

Andalusite

1200 1000

800

600

400

200

Raman shift (cm"'') Figure 1 Micro-Raman spectra of Al2Si05 polymorphs (Mernagh and Liu, 1991a).

cases. They can, however, be easily distinguished by their characteristic Raman spectra, as shown in Fig. 1 (lishi et at., 1979; McMillan and Piriou, 1982; Salje and Wernecke, 1982; Mernagh and Liu, 1991a). In contrast, it is usually difficult to distinguish the Ti02 polymorphs (brookite, rutile and anatase) using optical microscopy. However, the differences in the symmetry of the crystals result in completely different Raman spectra, which permit their unambiguous identification by micro-Raman spectroscopy (Beny et al., 1989). The identification of high-pressure polymorphs of Si02 has played a significant role in elucidating the P-T histories of natural and synthetic minerals subjected to high static or dynamic pressures. For example, the identification of coesite in a clinopyroxene from Norwegian eclogitic rocks in the Caledonides (Fig. 2) demonstrated that the rock formation pressure was at least 30kbar (Smith, 1984). This observation has had a profound impact on the controversial origin of eclogites in Norway. Boyer et al. (1985) have also used micro-Raman spectroscopy to study coesites in a range of natural eclogites, and to identify sub-microscopic grains of the low-pressure Si02 polymorph, quartz, presumably formed by reversion of the highpressure sample. Gillet et al. (1984) used micro-Raman spectroscopy in conjunction with TEM techniques to identify coesite inclusions within pyrope grains in metasedimentary rocks from subducted continental crust, and proposed an elastic model to explain the preservation of the high-pressure

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|521 CS

>1-

C/)

zLU

12 2

< DC

178 CS 151 1

a^ 118 <^^

271 CS 1

467

206 ICS+QZ

vUvw.^ JL

100

200

356 CS^QZ 1

1

300 400 Raman shift (cm-i)

1

1

1

500

1

1

600

Figure 2 Micro-Raman spectrum of coesite relic crystal as inclusions in clinopyroxene. CS, peak of coesite; QZ, peak of quartz (Smith, 1984).

Si02 phase within the garnet matrix. This model allowed constraints to be placed on the crystallization conditions and the subsequent P-T history of the pyrope-coesite assemblage. Calcium carbonate is found in two polymorphic forms, calcite and aragonite, the high-pressure polymorph. Gillet and Goffe (1988) demonstrated the utihty of micro-Raman spectroscopy for rapid and unambiguous identification of aragonite in thin sections from metamorphic terrains in the Alps (Vanoise, France). Le Cleac'h and Gillet (1990) also used micro-Raman techniques to characterize lawsonite, an important petrogenetic indicator, in thin sections of blueschist facies rocks from the Western Alps. Malezieux et al. (1983) demonstrated the utility of micro-Raman spectroscopy for characterization of the Cr/(Cr + Al) ratio in a series of natural chromites, calibrated from micro-Raman studies of synthetic chromite phases (Cervelle et al., 1984). The O—H stretching vibrations of amphiboles and micas give rise to a series of sharp peaks in the 3500-3700 cm"^ region. These features have been related to different Mi and M3 site occupancies, and powder-infrared studies have been employed to characterize the cation distribution. Various authors (Burns and Strens, 1966; Wang et a/., 1988a,b) have used micro-Raman spectroscopy to observe the O—H stretching peaks in a series of natural amphiboles. This technique has the advantage over powder-IR methods in that it is nondestructive, thus samples of the hydrous mineral can be studied directly in the form of thin sections.

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2. Shocked Phases and Meteorites The regions or phases of interest in these samples are often of ^xm size, and, because of their rarity, or in the case of laboratory-shocked samples, the difficulty in their preparation, it is important that they be studied with the use of a nondestructive method. The micro-Raman technique is ideal for such studies. Hemley et al. (1986b) and Mao et al. (1987) have used micro-Raman spectroscopy to study shocked quartz samples within the Coconino sandstone from Meteor Crater, Arizona. Both high-pressure phases of Si02, coesite and stishovite, were observed, confirming the occurrence of an impact event. In a similar recent study Halvorson and McHone (1992) observed Raman peaks characteristic of coesite in a matrix adjacent to a pseudotachylite vein in a quartzite from the Vredefort dome in South Africa, confirming the shock origin of the dome, associated with extraterrestrial impact. Boyer and Velde (1986) studied a series of naturally shocked quartz samples from the Ries (Germany) impact crater, and observed a high degree of vitrification. In a recent study of laboratory-shocked quartz samples, McMillan et al. (1992a) used the micro-Raman technique to investigate the onset of glass formation with increasing peak pressure in the shock experiments. In this work it was found that the appearance of diaplectic glass was accompanied by negative pressure shifts of the Raman lines of the crystalHne quartz matrix, due to the appHed tension (Fig. 3). For other shocked samples, the micro-Raman and electron microprobe techniques were combined (Velde and Boyer, 1985) in a structural and compositional investigation of naturally shocked microcline from an impact structure in Canada (Fig. 4). This study documented changes in the feldspar structure with shock, and permitted unambiguous identification of the diaplectic glass in the shocked samples to be made. This diaplectic glass had a different Raman spectrum from those of thermally produced glasses. In addition, the electron microprobe study demonstrated progressive changes in the composition of the diaplectic glass with increasing degree of shock deformation. Boyer et al. (1988a) carried out a similar micro-Raman analysis of laboratory-shocked albite samples, and Velde et al. (1989) and Heymann and Horz (1990) used the micro-Raman technique to investigate the formation of diaplectic glass in a series of plagioclase samples experimentally shocked to peak pressures above 50 GPa. The micro-Raman technique has also been invaluable in the analysis of fxm-sized phases in extraterrestrial materials. Boyer et al. (1988b) used this method to identify three polymorphs of Si02 (cristobalite, quartz, and most probably, tridymite) in a carbonaceous chondrite from La Reunion. Carbon has often been found in the form of SiC in chondrites, such as the Murchison CM2 carbonaceous chondrite. The micro-Raman spectra of SiC crystals with sizes ranging from a few luim to a maximum of 26 ixm permitted the identification of both cubic SiC and noncubic polytypes, as shown in Fig.

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Figure 3 Effects of shock pressure on the micro-Raman spectrum of quartz showing a negative pressure shift and weak broad bands near 500 and 600 cm"\ These broad bands are due to diaplectic glass (McMillan et al., 1992b).

5 (Virag et al, 1992). The comparison of the Raman spectra with isotope data obtained with an ion microprobe, suggests that all SiC grains with anomalous isotopic compositions have a cubic structure, while isotopically normal grains have noncubic ones. Most cubic SiC grains showed a weak, broad LO band, indicating an imperfect structure, which reflects conditions of formation or subsequent irradiation of the sample. McMillan et al (1989) used micro-Raman spectroscopy to study a natural majorite garnet, a potentially important phase within the Earth's mantle, which is present within the Catherwood meteorite.

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Figure 4 Micro-Raman spectra of artificially shocked anorthite (a) and albite (b) and naturally shocked microcline (c). In (c), M is unshocked microcline and spectra (1) to (4) refer to different points within the shocked sample. The broad bands correspond to the diaplectic glass; (a) and (b) are from Velde et al. (1989). Curve (c) is from Velde and Boyer (1985).

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Raman shift (cm-'') Figure 5 Micro-Raman spectra obtained from different SiC grains of the Murchison CM2. (a) Cubic lattice structure indicated by the single, transverse-optical (TO) peak; and the longitudinal optical (LO) peak. (b) Noncubic structure indicated by the splitting of the TO mode into three peaks.

Earth, Planetary and Environmental Sciences 299 3. Mineral Inclusions Micro-Raman spectroscopy is particularly useful for studying mineral phases included within another host mineral, as already described above for coesite inclusions. Recently, diamond inclusions were found in garnets in metamorphic rocks from the Kokchetav massif in northern Kazakhstan, FSU (Sobolev and Shatsky, 1990). Micro-Raman spectroscopy was one of the techniques used for the unambiguous identification of diamond crystals included in zircons or in unzoned garnets. This result proved that metamorphic pressures reached at least 40 kbar in these rocks, for temperatures in the 900-1000°C range. These P-T constraints have important impUcations for regional tectonic models, which must be accounted for by burial to 100 km depths, followed by return to the surface. Results of a similar micro-Raman study on diamond inclusions in garnets from the Anhui Province, China, have recently been reported (Shutong et al., 1992). In another study, Malezieux (1990) used the micro-Raman technique for the unambiguous identification of garnet, which is close to pyrope in composition, exsolving from clinopyroxene in natural mineral grains.

B. Phase Identification in the Environmental Sciences In this particular application the micro-Raman spectroscopic measurements are performed on single microparticles with masses as small as 10~^^ g. These samples are deposited on a suitable substrate, commonly either high-purity sapphire or lithium fluoride of single-crystal quality (Etz et al., 1911, 1978). The use of other substrates and the details of sample preparation, including the necessary precautions to prevent their deterioration, are discussed by Etz et al (1977) and Etz (1987). 1. Aerosols It is of major current concern to know if it were possible to alter the Earth's atmospheric chemistry and chmate by the injection of particulate material into the atmosphere (Charlson and Wigley, 1994). The South Pole, which is isolated from any major source of particles, is an ideal place to carry out such determinations. Analyses by SEM have shown high sulfur concentrations in virtually all of the particles found there. Supplemental investigations with the use of the micro-Raman technique have allowed the unambiguous identification of (NH4)2S04, as the principal sulfur-bearing phase present (Cunningham et al., 1979). The identification of marine aerosols is also very important for understanding the supply of matter to the continent by wind action. Such marine aerosols

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have been collected in clean marine air at the Enewetak atoll (Goypiron et al., 1982). The size of the particles investigated ranged from about 2 to 30 (xm, and calcite, gypsum, and orthoclase were identified by micro-Raman spectroscopy. In addition, a fatty acid has been recognized on a gypsum particle. A fibrous peptide, probably of the myosin or a-keratin type, has also been detected, although the identification of amino acids is not yet straightforward (Goypiron et al., 1982). 2. Airborne Urban Particles and Pollution Studies Airborne particulate dusts exhibit an enormous variety of types and compositions; thus, their analysis presents a major challenge. Several micro-Raman studies have been carried out on samples of St Louis, Missouri (USA) air dusts. Particles of calcite, anhydrite, NaN03, and V2O5 have been identified (Etz et al., 1977). In addition, many of these particles exhibit a Raman spectrum typical of poorly crystallized carbon (Blaha et al., 1978). The Raman spectrum of this carbon was independent of the irradiance level on some particles, and was attributed to soot. In contrast, on other samples the Raman signal appeared only at high irradiance levels. In laboratory studies, it was found that coating anhydrite crystals by Az-hexadecane produced Raman spectra of poorly crystallized carbon only under high irradiance levels. This result suggests that some particles are coated by organic compounds which can be transformed into carbon under laser irradiation (Blaha et al., 1978). These authors called attention to the need for a study of such samples at different irradiances in order to identify and distinguish intrinsic amorphous or poorly crystallized carbon, and laserinduced carbons. The advent of micro-Raman instruments with multichannel

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Figure 6 Micro-Raman spectrum of a particulate (V2O5) of oil-fired, power-plant origin collected on a sapphire substrate (Etz et al., 1978).

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detection, as well as Fourier transform instruments, which permit much lower excitation power, will be of major importance in such investigations. Oil-fired plants are known to be an important source of particulates in the atmosphere. The characterization of these particles is essential to evaluate their impact on the environment; thus, micro-Raman spectroscopy can play an important role in this work. As already noted, vanadium pentoxide particles have been identified (Fig. 6), from their characteristic Raman spectrum, to be the molecular form of vanadium present in the atmosphere (Etz et al., 1978). Generated poly dispersed sulfuric aerosols (Etz et al., 1977), prepared micro-particles of organic pollutants such as polynuclear hydrocarbons (Etz et al., 1979) and pesticides (Etz et al., 1978) can all be identified by the micro-Raman technique, demonstrating its analytical potential in environmental studies.

C. Establishment of Mineralogical Phase Diagrams

One of the conditions necessary for the use of observed mineral assemblages to characterize P-T conditions of rock formation, and their subsequent P-T history, is the existence of appropriate phase diagrams for the mineral systems of interest. These diagrams are either determined by direct measurement, with the methods of experimental petrology, or they can be calculated, if the thermodynamic properties of the mineral phases are known. These data are particularly important in determining the mineralogy of the deep Earth and other planetary interiors, where samples are scarce or impossible to obtain. In particular, recent developments in multiple-anvil and diamond-cell techniques have revolutionized ultra-high-pressure, high-temperature syntheses and phase-equilibrium experiments for exploring the mineralogy of the Earth's mantle and core. 1. Structural Characterization of Phases by High-pressure, High-temperature Experiments Micro-Raman spectroscopy has played an important role in the identification and structural characterization of the |xm-sized phases which are the usual result of high-pressure laboratory syntheses (Akaogi et al., 1984; Ross and McMillan, 1984; Ross et al, 1985; Guyot et al, 1986; Hemley et al, 1986a, 1987a, 1989b; Geisinger etal, 1987; Mao etal, 1987; McMillan and Akaogi, 1987; McMillan and Ross, 1987; Wilhams et al, 1987; Lienenweber et al, 1989; McMillan et al, 1989, 1991; Boehler and Chopelas, 1991; Fei et al, 1991; Graham et al, 1992; Zhang etal, 1993). Such syntheses are expensive and time-consuming; thus, the nondestructive, micro-Raman technique, which requires only a few grains of material, is ideally suited to their study.

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Of particular interest has been the characterization of stishovite, the high-pressure phase of Si02, the j8 and y high-pressure forms of Mg2Si04, and the ilmenite, garnet and perovskite phases of MgSi03. All of these substances are probably important mantle minerals; see Fig. 7 (Jeanloz and Thompson, 1983; Fei et al., 1990; Gasparik, 1990; Ringwood, 1991; Thompson, 1991). The identification and study of MgSiOa perovskite has been particularly crucial, because it appears to be a major constituent of the Earth's lower mantle (WilHams etal, 1989a; Hemley and Cohen, 1992). The identification of stishovite in natural samples is complicated by the introduction of impurities due to the extraction process. This problem only became apparent once the micro-Raman spectrum of a synthetic stishovite sample had been recorded (Hemley et al., 1986b; Hemley, 1987). The Raman spectrum of this phase is extremely interesting because one vibrational mode (Big) corresponds to the atomic displacements required to transform stishovite from the rutile into the calcium chloride structure, a potential post-stishovite phase in the mantle. This transition has recently been observed by in situ Raman spectroscopy at high pressure in the diamond-anvil cell (Kingma et al, 1993a). There is much interest both in stable, hydrous high-pressure phases, and in the presence of hydrous components in nominally anhydrous high-pressure minerals which may provide sources or sinks for water in the Earth's mantle (Bell and Rossman, 1992). Micro-Raman spectroscopy has been used to study the structure and nature of the OH sites in phase B (Mg23Si8042H6), an important high-pressure phase in the MgO-Si02-H20 system (Akaogi and Akimoto, 1986; Finger et al., 1989; McMillan et al, 1991). At the Mg2Si04 composition, the wadsleyite (j8-Mg2Si04) phase is specific, because two adjacent Si04 tetrahedra are linked by an SiOSi bridge which gives rise to a strong Raman peak at 723 cm~^ (Fig. 7b). Because the composition is that of an orthosilicate, formation of the SiOSi linkage in the high-pressure phase results in one oxygen per asymmetric unit being bound only to Mg atoms in an irregular five-fold coordinated site (Horiuchi and Samamoto, 1981) which could provide a potential site for protonation of the nominally anhydrous mineral (Smyth, 1987; Downs, 1989). This observation is an important one because j8-Mg2Si04 is Hkely to be a dominant constituent in the transition zone of the mantle at a depth of 400-500 km. Any hydration of this phase could significantly affect the mineralogy and seismic properties in this region. A synthetic sample of j8-Mg2Si04 was examined whose micro-Raman spectrum exhibited an asymmetric band at 3322 cm~^ due to the presence of OH groups within the structure (McMillan et al., 1991). Measurements of the absorbance using micro-infrared spectroscopy allowed an estimate to be made of the OH content of approximately 0.06% by weight (g OH per 100 g Mg2Si04). The mechanism of the olivine-wadsleyite-spinel transitions in the (Mg,Fe)2Si04 system is of particular interest because of its importance in

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Figure 7 Micro-Raman spectra of high-pressure phases in the Si02-MgO system, (a) Stishovite at atmospheric and high pressure (Hemley, 1987). (b) /3- and 7-Mg2Si04 (McMillan and Akaogi, 1987). (c) MgSi03 ilmenite (McMillan and Ross, 1987). (d) MgSi03 garnet, majorite (McMillan et al., 1989). (e) MgSi03 perovskite at atmospheric and high pressure (Hemley et al., 1989b).

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the mineralogy and rheology of the transition zone (Akaogi et al., 1989; Rubie, 1989; Guyot et al., 1991). In addition to carrying out in situ, high-pressure experiments of the forward transitions, Ming et al. (1991) have described the importance of studying the back-transformation of the high-pressure phases at ambient or low pressures. McMillan etal. (1991) used micro-Raman spectroscopy to study a sample of )8-Mg2Si04 which had been heated for several minutes at 580°C. They observed a spectrum similar to that of the low-pressure phase (forsterite, a-Mg2Si04), but exhibiting additional peaks in the 600-700 cm~"^ region. These features are characteristic of the presence of SiOSi linkages, indicating that the back-transformed sample contained some Si207 units, as in the high-pressure phase, in addition to isolated Si04 groups. Another interesting reversion study on a high-pressure mineral has been carried out by Durben and Wolf (1991) on the geophysically important perovskite phase of MgSi03. These authors used micro-Raman spectroscopy to study the variation of the Raman spectrum of this phase with increasing temperature at atmospheric pressure. In this investigation they found that above approximately 300°C MgSi03 perovskite begins to revert to a glass. In effect, this process puts the crystaUine lattice under compressive stress, resulting in shifts in the Raman bands towards higher frequencies. This observation places important constraints on high-temperature structural data measured on MgSi03 perovskite at atmospheric pressure. 2. High-pressure and High-temperature In Situ Studies Micro-Raman spectroscopy used in conjunction with the diamond-cell has been one of the most powerful techniques for characterizing Earth and planetary materials at ultrahigh (megabar) pressures, equivalent to those found deep within planetary interiors. The reason for this success has been due to the fact that such extreme pressures can be produced on very small samples (tens of |xm or less) in the laboratory; hence, micro-sampUng techniques are required to probe the material in situ under these conditions (Hemley et al, 1987a; Hemley and Porter, 1988). For phases which are likely to be present deep within the Earth, it is essential to characterize their structural and dynamic behavior at high pressures and temperatures. Micro-Raman spectroscopy is a technique of choice for such studies, especially for in situ work at high pressure with the diamond-anvil cell (Sharma et al., 1985; Hemley et al., 1987a; McMillan and Hofmeister, 1988; McMillan, 1989; Sharma, 1989). From available phase equilibrium data, high-pressure phases of Si02 are likely to be present within the mantle (Fei et al., 1990; Gasparik, 1990; Ringwood, 1991; Thompson, 1991). The best-characterized high-pressure phase of Si02 is stishovite, which has the rutile structure. The atomic displacements associated with the lowest-frequency (B^g) Raman mode of rutile-structured minerals correspond

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Figure 8 Variation of the Raman frequencies of stishovite with pressure (Hemley, 1987). to those required for a transition to the CaCl2 structure (Nagel and O'Keeffe, 1971). Hemley (1987), who used micro-Raman spectroscopy to investigate the vibrational spectrum of stishovite at pressures up to 33 GPa, observed that the frequency of the Big mode decreased ('softened') with increasing pressure, suggesting that a transition to a CaCVstructured, post-stishovite phase of Si02 might occur at pressures in the 100 GPa range (Fig. 8). This transition has now been observed by Kingma et al, (1993b). Hemley (1987) also observed evidence of a phase transition in coesite at 22-25 GPa. This work has recently been extended by Williams et al. (1993). Micro-Raman spectroscopy with the diamond-anvil cell has also been used to study the dynamics of MgSiOs perovskite up to 26 GPa (Hemley et al., 1989b). Gillet et al. (1993b) studied CaTi03 perovskite over a similar pressure range. Concerning the pressure dependence of lower-pressure phases which are stable in the crust and upper mantle, a number of workers have used Raman and micro-Raman techniques to investigate the vibrational behavior of forsterite and other olivines (Besson et al., 1982; Gillet et al., 1988, 1991; Chopelas, 1990; Liu and Mernagh, 1990), the Al2Si05 polymorphs (Mernagh

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and Liu, 1991a), several garnets (Mernagh and Liu, 1991b; Gillet et al., 1992), in addition to the extensive work which was carried out on a-quartz (Asell and Nicol, 1968; Dean et aL, 1982; Hemley, 1987; Jayaraman et al., 1987; WiUiams et al., 1993). There has also been much interest in the high-pressure structural and dynamic properties of carbonate minerals, in an effort to understand the phase stabihty of carbonates in the mantle, and the carbon budget and oxidation state of the deep Earth (Irving and Wylhe, 1975; Kushiro et al., 1975; Katsura and Ito, 1990; Blondy et al., 1991). Several micro-Raman investigations of carbonates have been carried out, at pressures in excess of 30 GPa (Gillet et al., 1988; Liu and Mernagh, 1990; Kraft et al., 1991; Biellman and Gillet, 1992; Gillet et al., 1993a). The study of calcite is particularly interesting, because two transitions to metastable forms of CaC03 (calcite-II and calcite-III) have been observed in the 1.4-2.0 GPa range (Fong and Nicol, 1971; Gillet et al., 1988; Hess and Ghose, 1988; Liu and Mernagh, 1990; Biellman and Gillet, 1992). In contrast, dolomite and magnesite show no evidence for any phase transitions up to the highest pressures employed (Kraft et al., 1991; Biellman and Gillet, 1992); nor does the stable, high-pressure aragonite phase of CaC03 exhibit such transitions. As noted earlier, micro-Raman spectroscopy is particularly adapted for in situ, high-temperature studies of minerals. This technique has been employed in such investigations of jxm-sized crystals of the high-pressure Si02 polymorphs coesite and stishovite (Gillet etal., 1990), and forsterite (Sharma, 1989; Gillet et al., 1991) and its germanate analogues (Gillet et al., 1989; Piquet et al., 1992), MgSiOs, CaGeOs and CaTi03 perovskites (Wolf et al., 1990a; Durben and Wolf, 1991; Durben etal., 1991; Gillet etal., 1993), and Ca-Mg carbonates (Gillet et al., 1993a). Sharma (1989) has also presented in situ, high-temperature, micro-Raman data for several polymorphs of MgSi03 enstatite. These last spectra are interesting in that they were obtained with a time-resolved technique which was used to eliminate the blackbody radiation background. These high-temperature observations are particularly important for exploring the intrinsic anharmonicity of vibrational modes, which can have an important effect on the high-temperature thermodynamic properties of these systems (Gillet et al., 1989a, 1990, 1991; Piquet et al., 1992). In particular, the work on the silicate and germanate oHvines has revealed some interesting indications of dynamic disorder at high temperatures which can be associated with a rapid increase in the mineral heat capacity just below the melting point, termed 'pre-melting' by Richet and Piquet (1991). Relatively little work has been done on rock-forming minerals under combined high-pressure, high-temperature conditions, although research is currently underway on this important topic in several laboratories. With the use of conventional (resistive) heating, Kraft et al. (1991) used micro-Raman spectroscopy with a diamond-anvil cell to investigate the vibrational spectrum

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of dolomite at pressures up to 11.5 GPa and temperatures to 550 K. In a similar study using conventional (macro-) Raman spectroscopy, Arashi (1987) investigated the monoclinic-orthorhombic phase transition in the important ceramic Zr02 under high P-T conditions. Most recently, Gillet et al. (1993b) have used micro-Raman spectroscopy in a diamond-anvil cell heated with a CO2 laser to follow the phase changes in CaTi03 perovskite to pressures and temperatures of 12 GPa and 1600 K, respectively. In addition to the intrinsic value of the micro-Raman data for identification and structural characterization of mineral phases, the vibrational data are essential for testing theoretical models, both empirical and ab initio, of their lattice-dynamical properties. These calculations are particularly important for understanding the phase stability and structural behavior of minerals at high temperatures and pressures which are often well out of the range of experimental measurements (Bukowinski and Wolf, 1986; Cohen etal., 1987; Hemley et al, 1987b, 1989b; Price et al, 1987; Wolf and Bubowinski, 1987; Wall and Price, 1988). Micro-Raman spectroscopy has also played an important role in establishing a basis for similar calculations for low-pressure mineral phases. For example, Sato and McMillan (1987) used the microRaman technique to obtain vibrational spectra of fxm-sized grains of isotopically substituted (^^Si-^^Si and ^^O-^^O) quartz (Fig. 9). These data were then used to test the results of a lattice-vibrational calculation on a-quartz, with the use of valence force constants derived from ab initio cluster calculations (McMillan and Hess, 1990). Measurements performed on BeO show that the wurtzite structure of the mineral (bromellite) is stable to at least 40 GPa (at 300 K), in agreement with theoretical calculations (Jephcoat et al, 1988).

3. Calculation of Thermodynamic

Properties

Because the lattice vibrations provide the primary sink for thermal energy in crystal structures, a knowledge of the complete vibrational spectrum, usually expressed as the phonon (vibrational) density of states [g(a>)], permits a calculation of the vibrational heat capacity Cy{T), and associated thermodynamic properties (Salje and Viswanathan, 1976; Kieffer, 1979a,b,c, 1980, 1982; Salje and Wernecke, 1982). In general, the total, vibrational density-of-states function is not known, and must be modeled from available experimental data. Kieffer (1979a,b,c, 1980, 1982) and Salje and Viswanathan (1976) have developed methods for constructing such models of the g{(o) functions with the use of information from Raman and infrared spectra, as well as elastic constant measurements. Heat capacity calculations have been carried out with this method for a wide variety of important rock-forming minerals, including high-pressure phases (Kieffer, 1979a,b,c, 1980, 1982; Akaogi etal, 1984; McMillan and Ross, 1987; Gillet etal, 1989b,

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Figure 9 Raman spectra of the isotopic species of a-quartz, Si02, ^^Si02 and Si^^02 (Sato and McMillan, 1987).

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MODEL I

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Figure 10 Calculation of the heat capacities of forsterite. (a) Models of the density of states consistent with the spectroscopic data (optical modes). The values shown in the boxes are the number of modes in that continuum, (b) Harmonic and anharmonic values of Q calculated with the densities of states shown in (a). D.P. represents the Dulong-Petit limit (Gillet et a/., 1991).

1990, 1991; Fei et al, 1990; Madon et ai, 1991; Hofmeister and Chopelas, 1991; Hofmeister and Ito, 1992). In these works the micro-Raman technique was essential to obtain the spectra of the iJim-sized particles which were available, especially of the synthetic, high-pressure phases, and to assign the observed spectra reliably to the phase of interest. Gillet et al. (1989a, 1990, 1991) have recently made a significant advance in the application of such vibrational heat capacity calculations - especially in the high-temperature limit - by expHcitly considering the anharmonicity of the vibrational modes (Fig. 10). For this calculation, the temperature- and pressure-induced shifts of the vibrational frequencies are measured separately and used to obtain the intrinsic mode-anharmonicity parameters for use in the heat capacity calculation. The anharmonicity is shown to have a large effect on the calculated heat capacities at high temperature, as they are significantly higher than those calculated with the use of the harmonic model. The effects on thermal expansion have also been explored by Hemley et al. (1991). In all of this work micro-Raman spectroscopy proved to be a convenient tool for the study of these high-temperature and high-pressure vibrational properties.

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D. Phase Transitions in Minerals 1. Displacive Phase Transitions One of the classic applications of Raman spectroscopy in solid-state physics and chemistry has resulted from the study of displacive phase transitions through the observation of soft modes. A soft mode is an anharmonic vibration whose atomic displacements indicate the displacive changes associated with the phase transition. They can be driven thermally, by increasing the pressure, or by compositional changes (Raman and Nedungadi, 1940; Cochran, 1960, 1961; Scott, 1974; Samara and Peercy, 1981; Jayaraman, 1983; Ferraro, 1984; Wang 1984; Hemley et al, 1987a; Wong, 1987). The first Raman investigation of temperature-induced mode softening was carried out for the a-j8 quartz transition by Raman and Nedungadi (1940), who observed that the broad, 207 cm~^ band decreased rapidly in frequency with increasing temperature, to disappear at the a-j8 phase transition temperature (Fig. 11). They proposed that the atomic displacements associated with the 207 cm ~^ band might mimic the a-)8 displacive phase transition, which involves a co-operative rotatory movement of Si and O about the three-fold screw axes in the quartz structure. There have since been many experimental studies of the lattice dynamics of quartz through its a-/3 phase transition with the use of a variety of spectroscopic, thermodynamic and structural techniques; this basic picture is generally confirmed (Shapiro et al., 1961 \ Scott, 1968; Axe and Shirane, 1970; HochH and Scott, 1971; lishi, 1978), although the details of the phase transition near the transition temperature are considerably more complex than originally thought. For example, it has been shown that the a-)8 quartz phase transition does not proceed directly, but that the a and j8 phases are related by a series of incommensurate phases (DoUno, 1986). A second complication arises with the nature of the high-temperature phase, which may have either a dynamic or statistically averaged structure above the phase transition temperature (Dohno et al, 1983; McMillan and Hess, 1990). A second class of geophysically important compounds to which soft-mode Raman spectroscopy has been applied is perovskite; these compounds exhibit a rich series of phases with different degrees of structural distortion, starting from the ideal cubic structure. These phases are connected by displacive phase transitions (Cochran and Zia, 1968; Lockwood and Torrie, 1974). The earliest Raman work was carried out on SrTi03 perovskite, which showed a cubic-tetragonal phase transition at 110 K. The cubic perovskite phase has no allowed, first-order Raman bands; thus, the Raman spectrum is characterized by broad bands due to second-order vibrational transitions (Nilsen and Skinner, 1986). The transition to the tetragonal phase is marked by the appearance of a set of sharp Raman peaks, which are the allowed first-order Raman spectrum of the lower-symmetry phase (Nilsen and Skinner, 1986;

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Ai 215 cm"* (207)

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Figure 11 The a-p displacive phase transition in quartz. (A) displacement vectors for the 207 cm~^ mode of a-quartz, and for the a-jS quartz structural displacement (McMillan, 1985). (B) High-temperature behavior of the 207 cm~^ mode (Shapiro et al., 1967). (C) Plot of the square of the Raman shift (v^) versus T-T^ for the 207 and 147 cm~^ modes, as shown in (b) and (c) of part A. Fleury et al., 1968). There is currently intense interest in the structure and dynamics of MgSi03 perovskite. Recent micro-Raman studies at high pressure and room temperature (Hemley et al., 1989b, 1990), and atmospheric pressure at high temperature (Wolf et al., 1990a; Durben and Wolf, 1991), have suggested that this is a major constituent phase of the Earth's mantle (Jeanloz and Thompson, 1983). No evidence has been found for such second-order displacive transitions in MgSi03 perovskite, although a firstorder phase transition appears to have been observed (Wang et al., 1991b). Gillet et al. (1993b) have recently used micro-Raman spectroscopy to investigate the behavior of CaTiOs perovskite at high pressure (up to 21GPa) and high temperature (up to 1450 K). They found evidence for the orthorhombic-tetragonal-cubic phase transitions suggested by both calorimetry and X-ray diffraction.

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There have been several Raman and micro-Raman studies on the influence of pressure on compounds with the rutile structure. There is a known high-pressure form of titanium dioxide (Ti02-II), which was originally suggested to have the a-Pb02 structure (BendeHani et al., 1966). Nicol and Fong (1971) and Samara and Peercy (1981) observed a softening of the lowest frequency (Big) mode of Ti02 with pressure using in situ Raman measurements. The displacements associated with this mode would lead to a transition to the CaCl2 structure, if the mode became dynamically unstable, suggesting that this structure might be possible for the high-pressure phase (Nagel and O'Keeffe, 1971). However, it has been shown (Mammone et at., 1980) that the Raman spectrum of Ti02-II is inconsistent with the CaCl2 structure. The latter authors concluded that the high-pressure phase has, in fact, the a-Pb02 structure. The same type of Big-mode softening has been observed in Sn02 by Peercy and Morosin (1973), and for Si02 by Hemley (1987), and by Kingma et al. (1993a,b).

2. Order-Disorder

Transitions

(a) Graphite and related compounds Raman spectroscopy has also been used in the analysis of order-disorder processes relevant to mineralogy and geochemistry. The study of graphiterelated carbonaceous compounds provides one of the simplest applications. Graphite is formed mainly in the continental crust, either from precipitation from a fluid phase, or, more commonly, from some organic precursor embedded in sediments under conditions of increasing temperature and pressure during burial to metamorphic conditions. This process is termed 'graphitization'. The degree of disorder in the poorly crystallized graphitic material can be an important petrogenetic indicator. Raman spectroscopy has been adopted as a useful tool for quantitative characterization of the degree of order in graphites. Because the carbonaceous particles can be very small, of the order of only a few |xm, the Raman microprobe technique is ideally suited for the study of natural and synthetic samples undergoing this graphitization process (Beny-Bassez and Rouzaud, 1985; Pasteris, 1988; Pasteris and Wopenka, 1991). Micro-Raman spectroscopy is also of interest because it is an in situ, nondestructive technique that does not require any extraction procedure during which the organization state of the material might be altered. In addition, the spatial scale probed by micro-Raman spectroscopy is of the order of that probed by medium- to high-resolution electron microscopy; thus, the onset of ordering can be examined in the samples (McMillan, 1984a). Perfectly ordered graphite has P63/mmc = Z)6h space-group symmetry. There are two Raman-active vibrational modes with E2g symmetry: one at

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42cm~^ which can be described as a ghding of adjacent aromatic planes (E2g^) with respect to each other, and the second at 1575 cm" ^ (E2g2)' corresponding approximately to C—C stretching within the hexagonal layers (Tuistra and Koenig, 1970; Song et al., 1976). An overtone vibration is also observed near 2700 cm~^. The graphite structure can be disordered in several ways: by relative rotation of adjacent layers, by puckering the planar sheets, or by disorder in the interlayer spacing (Rouzaud et al., 1983; Buseck and Bo-Jun, 1985). Any disorder has a marked effect on the Raman spectrum. With increasing disorder, the E2g2 band broadens and moves to higher frequency, and a new band appears at 1350 cm"^. The relative intensity of this additional band has been correlated with a structural 'correlation length' (La), determined from X-ray data, which corresponds to the average interatomic layer separation in the disordered structure (Tuistra and Koenig, 1970; Lespade et al., 1982, 1984). These effects have been explained by considering the phonon dispersion curves of perfectly ordered graphite (Lespade et al., 1982). If the crystal is uniformly disordered, this interpretation is equivalent to a consideration of a larger unit cell (or smaller Brillouin zone), so that the vibrational density of states makes a more important contribution to the Raman spectrum. The disordered structure is often identified by the appearance of the so-called Boson band at low frequencies. Micro-Raman data have been correlated with observations by optical and transmission electron microscopy on graphitizable and nongraphitizable reference-carbon series, as a function of heat treatment by Beny-Bassez and Rouzaud (1985); see Fig. 12a. In this work the graphitization process was monitored by the L^ value, which was directly measured from lattice fringes and dark-field micrographs obtained by electron microscopy, as well as the intensity of the 1350 cm~^ Raman band relative to that near 1600 cm~^. In the series of oxygen-poor, graphitizable anthracene cokes, the graphitization is characterized by a three-step process (Fig. 12b). The end-product of this heat-treatment is highly ordered graphite, obtained at 2700°C, accompanied by complete disappearance of the 1350 cm~^ default band. In contrast, nongraphitizable carbon heated at atmospheric pressure always exhibits the 1350 cm~^ defect band, even for samples heated to 3000''C. The role of pressure during the graphitization process has been demonstrated experimentally by Beny et al. (1986), who rapidly transformed nongraphitizable cokes into graphite at 1800°C and 5 kbar pressure. Because the degree of order in these samples is sensitive to temperature and pressure, and can be monitored with the use of Raman spectroscopy, the micro-Raman spectra of natural, graphitized samples can provide a useful metamorphic indicator. First-order micro-Raman spectra of eight carbonaceous samples from metasediments show a progressive decrease in the width of the £2g2 band, a decrease in its frequency to 1575 cm"^, and a decrease of the intensity ratio 3(1360 cm~^)/3(1580cm~^) from 1.2

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0

10^/L

2900°C 80

2500°C 70

J.

2000°C 1800°C 1500°C

60

^^^^^^^

1300°C

50

Vf^^.w/'''f^

***^

""N^^iv* 800°C

40 30 A

^^^j

% H 600°C

/ Vw^^^'^v. . ^

20 A

10

^''--- SEMI-COKE 1600

1400

1200

Raman shift (cm-i)

10

20 30

40 50

60

-^1350

Figure 12 (a) Raman spectra of anthracene cokes heat-treated at different temperatures, (b) 10^/La versus 'S^i35o- The correlation length L,^ was determined from X-ray data; 51350 is the ratio of the integrated intensities of the 1350 and 1600 cm~^ bands (Beny-Bassez and Rouzaud, 1985). to 0.1 on passing from the low-grade metamorphic (prehnite-pumpellyite) to the high-grade metamorphic (staurolite) environments (Beny and JehHcka, 1991). In contrast, breaks in the evolution of the Raman spectra of graphitic carbons, documented by changes in the intensity ratio 3 (1350 cm~^)/3 (1580cm~^) of a series of metapelites, seem to be correlated with changes in silicate mineralogy (Pasteris and Wopenka, 1991). These authors suggested that the release of fluid, accompanied by locahzed stresses on carbonaceous grains due to changes in grain size, might produce changes in carbon crystallinity. These studies indicate that a great number of parameters control the graphitization process: the nature of the organic precursor, the pressure and any deviative stresses, the mineral reactions, the precipitation of graphite directly from the fluids, and perhaps other factors. This result opens up a new area in the investigation of metamorphic terrains with the use of a combination of micro-Raman spectroscopy and highresolution microscopy to analyze the carbonaceous species present. In a different type of study, the disordering of an initiafly perfectly ordered graphite sample in an alteration zone associated with a uranium deposit was monitored by micro-Raman spectroscopy (both first- and second-order

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Raman spectra), high-resolution TEM and X-ray diffraction (Wang et at., 1989). It is worth noting that the X-ray diffraction technique was not sensitive enough to monitor the increase in disorder of the graphite. In the micro-Raman spectra, the increase in disorder is marked by a frequency increase of the E2g2 band, an increase in its FWHM, and by an increase in the intensity ratio 3(1350 cm" V3(1580cm~^). This result is consistent with a decrease in the L^ parameter from 1000 to 50 A for the most altered graphite (from 100 to 5nm). (b) Silicates and other minerals McMillan et al. (1984b) investigated the Raman spectra of a series of well-characterized synthetic cordierite samples with differing degrees of Al-Si order. They found systematic changes in the spectra, including narrowing and splitting of peaks, with increasing Al-Si order. These changes coincided with structural changes at distances of the order of 100 A, as determined by electron microscopy. These changes appeared well before any transformation was apparent with optical microscopy or X-ray diffraction. Putnis (1980a,b) and Clemens et al. (1987) used micro-Raman spectroscopy, combined with high-temperature solution calorimetry, X-ray diffraction and high-resolution TEM, to investigate Al-Si and stacking disorder in phlogopite. McMillan et al. (1989) combined micro-Raman and infrared spectroscopy with ^^Al NMR spectroscopy to study cation-site ordering in high-pressure garnets along the Mg3Al2Si30i2-Mg4Si40i2 join. The disordering of ions over the A and B sites in spinels results in the appearance of additional Raman peaks due to a reduction in local symmetry (Fraas et al, 1973; McMillan and Hofmeister, 1988; McMillan et al, 1989). Malezieux et al. (1983) used the micro-Raman technique to investigate a series of natural spinels with different degrees of structural order. Cynn et al (1991) employed Raman spectroscopy to determine the cation ordering in MgAl204 spinel in situ at high temperatures, while Hofmeister and Chopelas (1991) studied garnet solid solutions. Salje (1985) has pioneered the apphcation of Landau's theory to the problem of Al-Si disorder in alkaU feldspars, and has described a method for using the temperature dependence of Raman-vibrational intensities as a parameter for quantifying both Al-Si and alkali-cation ordering in these phases (Salje, 1986). This technique will certainly prove to be a powerful one in future studies of ordering in aluminosiUcate and other minerals. 3. Pressure-induced

Amorphization

One of the more novel appHcations of micro-Raman spectroscopy is its use in the study of amorphization phenomena, specifically in pressure-induced

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amorphization. Raman scattering has been particularly useful in this regard because vibrational measurements are sensitive to both long- and short-range order in materials; i.e. the vibrational frequencies are determined to a large measure by local bonding properties, whereas the number and symmetry of bands are dictated by the long-range order in the crystal. The changes in both are therefore useful indicators of the loss of order at different length scales in the materials, as these observations complement the results of more direct probes of crystallinity, such as X-ray diffraction. Following the discovery using Raman spectroscopy of this amorphization phenomenon in ice (Mishima et al., 1984), the transformation was subsequently confirmed by Hemley et at. (1989a) with synchrotron radiation, which demonstrated the disappearance of the crystalline diffraction peaks, and by micro-Raman spectroscopy. This phenomenon was first observed in the Si02 polymorphs a-quartz and coesite at 25-35 GPa with the use of micro-Raman, diamondcell techniques (Hemley, 1987; Kingma et al., 1993b). In some recent studies of the transition in a-quartz Si02 and Ge02, micro-Raman spectroscopy has been combined with TEM to investigate the microstructural changes which accompany the amorphization process (Verhelst-Voorhees and Wolf, 1992; Wolf et al., 1992; Kingma et al., 1993b). Other minerals that have been studied by micro-Raman spectroscopy which are observed to undergo such pressure-induced crystal-amorphous transitions include cristobalite (Halvorson and Wolf, 1990; R. J. Hemley, unpubhshed), serpentine and portlandite, Ca(OH)2 (Meade etal., 1992) and Ge02 (Wolfed al., 1992), respectively. A number of minerals also undergo intermediate, metastable crystalline-crystalline transitions prior to amorphization. Examples include coesite (Hemley, 1987), cristobalite (Palmer er a/., 1992; Gratz etal., 1993), and serpentine and portlandite (Meade etal., 1992). In addition, there has been a growing number of observations of pressure-induced amorphization of related (e.g. mineral-like) materials with the use of micro-Raman, diamond-cell techniques (Fujii et al., 1985; Sankaran et al., 1988; Jayaraman et al., 1992; Serghiou and Hammack, 1992). Finally, micro-Raman measurements have also been carried out at atmospheric pressure on mineral samples amorphized by shock compression in the laboratory (Velde et al, 1989; Clough et al., 1992; McMillan et al, 1992a).

E. Micro-Raman Studies of Condensed Gases Perhaps most important to planetary science, as well as to condensed matter physics, is the behavior of hydrogen under ultrahigh pressures. Specifically, the understanding of the nature of the theoretically predicted transition of solid hydrogen to high-pressure metallic states requires detailed information on the structural, dynamical, and electronic properties of the material at ultrahigh pressures (100 GPa) (see Mao and Hemley, 1992, for a review).

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The characterization of the soUd under these extreme conditions has reUed exclusively on spectroscopic techniques used in conjunction with the diamond cell. Among these investigations, vibrational micro-Raman spectroscopy has been particularly useful, and has provided unique information on the behavior of hydrogen to near 300 GPa. As such, these measurements provide an important complement to other techniques, including X-ray diffraction, optical absorption and reflection, and infrared spectroscopy. Raman excitations in solid hydrogen involve intramolecular vibrational transitions (vibrons), lattice-mode excitations (phonons), and rotational bands (rotons). Measurements of the vibron frequency (VQ = A\55cmr^) provide a sensitive probe of the state of bonding in the molecular solid. The first Raman studies carried out up to 60 GPa with a diamond-anvil cell demonstrated that the frequency of the Raman-active vibron decreases with pressure above 30 GPa (Sharma et al., 1980). Subsequent investigations with the use of beveled, diamond anvils showed that the negative pressure shift continues to at least 147 GPa (Mao et al., 1985) and to - 2 5 0 GPa, as shown in subsequent work performed at 77 K (Hemley and Mao, 1988). These measurements demonstrate that the molecular bond is stable, although weakened, at these pressures. Recent Raman and infrared measurements indicate that the negative pressure shift can be understood in terms of a dramatic increase in intermolecular coupUng with pressure (Brown and Daniels, 1992; Hanfland et aL, 1992; Loubeyre et al, 1992; Silvera et al., 1992). At the highest pressures (corresponding to vibron frequencies at 3725 cm~^) there is also evidence for resonance enhancement at visible wavelengths, consistent with changes in electronic properties at these pressures, as measured by direct optical methods (Mao and Hemley, 1989). The Raman measurements of the vibron have also been instrumental in the discovery of a phase transition in sohd hydrogen at 150 GPa and low temperatures (Hemley and Mao, 1988; Fig. 13). The transition is characterized by a major, discontinuous shift in the vibron frequency at —100 cm~^ at a temperature of 77 K. A large number of subsequent experiments have been performed to determine the extent to which the transition is associated with an orientational ordering, or a structural or electronic transition (Hemley and Mao, 1989, 1990; Lorenzana et aL, 1989, 1990; Hemley et aL, 1990). An electronic transition could include the metallization process itself or the formation of a localized (e.g. excitonic) state. Measurements as a function of temperature reveal that the discontinuity decreases with increasing temperature up to a triple point. Direct measurements as a function of temperature show that the triple point is at —130K (Hemley et aL, 1990). Recent work shows that an analogous behavior occurs in deuterium (Mao and Hemley, 1994). Bands in the low-frequency Raman spectrum of molecular hydrogen (<1000cm~^) arise from rotational [5o(0) and Si{0)] and lattice-mode

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1

Solid Hydrogen Raman Spectra 77 K

l / \

141 GPa

145 GPa wvw'W^YV^^^ ^^

^

-^.^...^y^^^^yK

155 GPa

160 GPa 1

3700

3900

1

1

4100

4300

Wavenumber (cm" ; (a) 4150 4100 4050 h 4000 h > 3950 CO 3900 h 3850 130

140

150 160 Pressure fGPa)

170

(b) Figure 13 (a) Raman spectra of solid hydrogen obtained with the use of a diamond-anvil cell, in the region of the k = 0 intramolecular stretching modes (vibrons) through the phase transformation at 77 K. (b) Pressure dependence of the two vibrons in the region of the phase transformation. The lines are weighted, least-squares fits to the data (Hemley and Mao, 1988).

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excitations (E2g phonon in the hep solid). Detailed low-frequency Raman measurements indicate that the underlying hexagonal structure persists to pressures above 100 GPa (Hemley et al., 1990). A gradual broadening of the rotational (librational) bands is observed in both isotopes with increasing pressure above 100 GPa, with no abrupt change across the 150 GPa transition. This result confirms the conclusion that the principal changes at the phase transition occur in the intramolecular interactions, while structural or orientational effects may be secondary. Further measurements at higher pressures reveal an extremely intense, low-frequency Raman band that appears abruptly above 150 GPa in samples of hydrogen observed in a diamond cell (Hemley and Mao, 1992). The band has an initial frequency near 250 cm~^ and disappears above a critical temperature. This new feature may be diagnostic of a new phase of diamond, an unusual diamond-hydrogen interaction, or a new state of hydrogen. A related system is solid N2, which has been studied in the diamond cell under pressure by LeSar et al. (1979) and Daniels et al. (1981). More recently, this solid was investigated at megabar pressures by Reichlin et al. (1985) to 130 GPa and by Bell et al (1986) and Mao et al. (1986) to 180 GPa. This work indicates that the molecular bonds remain intact to these pressures (at room temperature), rather than dissociating to form a nonmolecular (and perhaps metallic) soUd, as predicted by theory for this pressure range. One N—N stretching mode of nitrogen begins to decrease in frequency above approximately 60GPa (Reichlin et al., 1985). Although there is no direct evidence for pressure-induced dissociation of the molecular bonds, the Raman data provide an indication of a number of structural transitions within the molecular soHd over this pressure range (Buchsbaum et al., 1984; Schneider et al, 1992). Water is another very important planetary material. Walrafen et al. (1988) studied the evolution of hydrogen bonding in Hquid water at high pressures (to 3.3 GPa) and high temperatures (to 200°C) by Raman scattering with a diamond-anvil cell. The behavior of H20-ice under pressure, including its high-pressure, phase relations and structural changes, has been the subject of a number of micro-Raman studies (Hirsch and Holzapfel, 1986; Hemley et al, 1989a; Pruzan et al, 1992; Walrafen et al, 1982). Of particular note are the recent observations of the metastable, low-temperature transitions, including amorphization (Hemley et al, 1989a), and the determinations of the equilibrium phase diagram and the ice VII-VIII transition at pressures up to 51 GPa (Pruzan et al, 1992). In addition to H20-ice, other simple molecular solids known as planetary ices have been studied; these substances include CH4 (Fabre et al, 1982), CO2 (Olijnyk et al, 1989) and NH3 (Gauthier et al, 1987). Condensed oxygen has been investigated by Raman spectroscopy in the diamond cell up to 40 GPa (Nicol and Fong, 1971; Nicol et al, 1979; Syassen and Nicol, 1981). This material exhibits a number of phase transitions accompanied by

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color changes. There has also been some work at lower pressures on the solid phases of CH4 (Fabre et al., 1982). Recently, micro-Raman techniques have been used to study phase relations in the simple molecular materials representative of the interiors of the large outer planets and their satellites. Of particular interest has been the observation of new, high-pressure sohd phases in the He-N2 and H2-H2O systems (Vos et al, 1992, 1993). Other systems include H2-He (Loubeyre et at., 1985, 1991b), H2-Ne (Loubeyre et at., 1991a, 1992a) and O2-N2 (Baer and Nicol, 1989). The behavior of carbon at high pressures is important for understanding the genesis, stability, and possible phase transitions in diamond within the Earth's mantle. Diamond itself has been the subject of a number of micro-Raman investigations to 20-40GPa (Boppart et at., 1985; Hanfland and Syassen, 1985; Hanfland et al., 1985; Sharma et al, 1985; Aleksandrov et al., 1986). These studies confirm the high-pressure stability and singular high strength of this mineral over this pressure range. More recently, Mao and Hemley (1991) reported results of spatially resolved micro-Raman measurements on anvils in diamond-cell investigations at sample pressures up to 300 GPa and found evidence for structural transformations driven by the large nonhydrostatic stresses of the anvils under these conditions. Goncharov (1990) and Hanfland et al. (1989, 1990) studied the roomtemperature transformation of graphite into a new, high-pressure form at 15 GPa. Although the transition occurs within the stabiHty field of diamond, the structure of the phase is not that of cubic diamond. The spectrum resembles that of an amorphous form (Goncharov, 1990), and thus the material has a well-defined crystalline diffraction pattern (Hanfland et al., 1990).

IV. GEOCHEMISTRY A. Fluid Inclusions Many rock types, especially ore deposits, are formed in the presence of a fluid phase. The most spectacular surface evidence for the circulation of fluid phases at depth is provided by geysers, and by the 'black smokers' found at oceanic ridge sites, which precipitate sulfides. The composition of the fluid phase which was at equilibrium with rock-forming minerals at depth can be calculated in some cases, but only if the variance of the mineral assemblage is not too great, and if thermodynamic data are available for both the minerals and the fluid species. However, even if these conditions are fulfilled, the fluid flow can be channeled through quartz veins, as is the case in many hydro thermal ore deposits. The fluid is then not necessarily at equilibrium with the vein host rock, and therefore its composition is poorly constrained.

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However, relics of these paleo-fluid circulations can often be found in the form of intracrystalHne cavities, termed fluid inclusions. Their size is highly variable, from a few jxm in diameter to 100-200 jjim, but most lie in the 5-20 juim range. Fluid inclusions are constant-volume and -mass systems. Therefore, any quantitative use of fluid inclusions requires the determination of both the molar volume {V) and the composition {X) of the trapped fluid. A single crystal may trap fluids during its growth, leading to the formation of primary fluid inclusions. If that crystal is subsequently submitted to stress, it may develop cracks. Then the fluid phase, if present in the rock-pore space, can be preserved as a secondary fluid, after healing of the microcrack. Each microfracturing event, associated with one tectonic phase, may result in the formation of a new generation of fluid inclusion, each having its own V-X properties, as it was trapped under specific pressure-temperature conditions. Therefore, it is necessary to use techniques which give information on the V-X properties for individual inclusions in order to obtain reliable geochemical information. The primary technique used for the characterization of fluid inclusions has been, and still remains, microthermometry. This method involves measuring the temperature of phase transitions occurring upon slowly cooling and heating the sample over the temperature range - 1 8 0 to 600°C. The observation is carried out with the use of a variable-temperature stage, interfaced with an optical microscope (Poty et al., 1976). Therefore, fluid inclusion research requires a probe with the same spatial resolution as the visible Hght used for optical microscopy. This characteristic is provided by the Raman microprobe, which explains the success of this technique for fluid inclusion analysis. The sample used for microthermometric studies is a paraUel-cut plate of 150-500 |xm thickness; it is thus ideally suited to Raman analysis. The composition of inclusions studied is highly variable, and their components belong to one of the following types: hydrocarbons heavier than CH4, simple small molecular species in the C-O-H-N-S system, and monatomic or polyatomic ions. In addition, fluid inclusions can contain minerals which were trapped with the fluid, or which nucleated during cooUng after fluid trapping.

1. Cations and Anions in the Aqueous Phase Constituent ions found in fluid inclusions can be classified into two categories based on their Raman spectroscopic characteristics: polyatomic ionic species and monatomic ions. Polyatomic ions such as NOJ^, COs", H C O J , S04~, HSO^ and H S " exhibit Raman spectra due to their internal vibrational modes, and can be easily detected. The detection limit for S04~ has been

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HS04(Ai) O

O A

CO

z

11 11

QZ

LU

SOl-(Alg) o CO o^

/ \

z

1 HS04(Ai)

1 \

\

§ AQZ

< 1

1

1200

_i

1

1

1000 Raman shift (cm-i)

•

1

800

Figure 14 HSO4 and SO4 identified by micro-Raman spectroscopy in a fluid inclusion in the aqueous phase inside a quartz crystal (QZ) (Dubessy et al., 1992a). demonstrated to be 0.01 m o l P ^ (Rosasco and Roedder, 1979; Dubessy et al., 1982), and is likely to be the same for other, similar, molecular species. Phosphates have never been identified by micro-Raman spectroscopy in fluid inclusions, because the low solubility product of phosphate-bearing minerals, such as apatite, commonly fixes the phosphate concentration in geological fluids at much less than 0.01 molal. Nitrate has also never been detected because the low E^ values of geological fluids, which are not in contact with the atmosphere, make this species unstable under normal crustal conditions. At room temperature carbonate becomes a dominant species in H2O-CO2 fluids only at unrealistically high pH conditions (>10.5). The bicarbonate ion is a weak Raman scatter, and has not been detected in fluid inclusions. All of these factors contribute to the observation that the main polyatomic ions identified in fluid inclusions to date are only S04~ and HS~ (Rosasco and Roedder, 1979). The sulfate concentration in primary fluid inclusions from upper triassic evaporates has been measured by Dubessy et al. (1983). In this work it was shown that the sulfate concentration was too low to have resulted from simple evaporation of a modern sea-water. The authors suggested a supply of calcium in the evaporating basin, probably due to dolomitization of carbonates found on the southern border of the German basin near the Alps. More recently, the bisulfate ion has been identified in the aqueous phase of complex fluid inclusions containing a mixture of liquid N2 and CO2 in the volatile phase (Dubessy et al, 1992a; Fig. 14). A calibration of the intensity

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ratio of the two bands has allowed an estimation to be made of the concentration of an extremely acidic fluid, with pH around zero. This is the first time that such highly acid geological fluids have been found. The samples were collected from a quartz vein cross-cutting itabyrites in the Tron quadrilateral' (Brazil). This observation raises two interesting geochemical questions. Did the fluids result from the oxidation of pyrite? Do the oxidizing fluids represent leachates of sulfate-bearing evaporate rocks? Unfortunately, the strong lateritization of the vein host rocks obliterated all of the information which could have led to an explanation of these unusual fluids, among the most acidic geological fluids ever documented. Hydrated monatomic ions, such as Na"^, K"^, Ca^"^, Mg^"^ and Fe^^, give rise only to weak bands in the 350-600 cm~^ spectral range. They are assigned to vibrations of the cation relative to the oxygens of the water molecules of the inner hydration sphere (Brooker, 1986; Dubessy, 1986). However, these bands are not useful for the identification of the particular cations present in an inclusion because they are very weak and are usually obscured by Raman bands or any, even weak, luminescence of the host crystal. On cooHng, the cations (R) mentioned above, together with chloride, the dominant anion of most geological fluids, nucleate salt hydrates, which can be described by the general formula R^C1„./7H20. These salt hydrates have different structures depending on the nature of the hydrate-forming cation, and have characteristic Raman spectra. The nucleated hydrate can then be easily identified if a microthermometric stage is coupled with the microRaman spectrometer, allowing the collection of the Raman spectra at different temperatures. Raman spectra have been obtained for the following hydrates: NaC1.2H20, CaCl2.6H20, MgCl2.6H20, MgCl2.12H20, KCl.MgCl2.6H2O, FeCls. I2H2O (Dubessy et al., 1982), LiCl. 5H2O (Dubessy et al., 1992a) and probably also CaCl2.4H20 (Schiffries, 1990). The identification of different hydrated crystals at different temperatures permits, first, a choice of an appropriate simplified projection in a ternary system (H20-Salt 1-Salt 2) to be made, and second, the reconstruction of the Uquid-composition path to the disappearance of the last soHd phase. The latter can be used to give a semi-quantitative estimate of the fluid composition. An illustration of the method is provided by the analysis of fluid inclusions in a quartz sample from the Bushveld complex (Schiffries, 1990). A new class of Hquid-absent fluid inclusions, containing halite (NaCl), antarticite (CaCl2. 6H2O) and probably a polymorph of CaCl2. 4H2O in the presence of a vapour phase, was documented at room temperature (Schiffries, 1990; Fig. 15). Upon heating, antarticite melted incongruently at 29°C. More than 25 vol% of the cavity is filled by the hquid at 31°C. With increasing temperature, the remaining hydrate (CaCl2.4H20) melted between 31 and 38°C, leaving as a soUd phase only halite, which disappeared at approximately 200°C. This phase behavior upon heating implies a high concentration of

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3100

3300

3500

3700

Raman shift (cm-i) Figure 15 Raman spectra of the OH stretching mode region of antarticite daughter crystals with decreasing temperature (Schiffries, 1990).

dissolved solids (>52wt%) and a high Ca/Na ratio. Grishina et al. (1992) studied another series of liquid-absent inclusions, trapped inside a metamorphic evaporitic halite, which exhibits similar behavior upon heating. As emphasized by Schiffries (1990), aqueous fluid inclusions that do not contain a liquid phase at room temperature may commonly be overlooked, or misinterpreted as mineral inclusions, although they are relics of highly saline fluids. Therefore, the investigation of salt hydrates by micro-Raman spectroscopy, coupled with microthermometric analysis, is essential for the identification of calcium-rich fluids. The chloride anion is a hydrogen-breaker; thus it strongly modifies the shape of the O—H stretching band of the aqueous phase (Walrafen, 1964, 1966). In contrast, cations have been found to have little effect on the frequency at maximum intensity, or the shape of the O—H stretching band. This observation has been recently used as a tool for the determination of the concentration of chloride in fluid inclusions (Mernagh and Wilde, 1989). These authors experimentally determined a 'skewing parameter' of the O—H stretching band with the use of synthetic-fluid inclusions in the NaCl, KCl, MgCl2 and CaCl2 systems. Based on the fact that the Raman spectra of aqueous solutions of different halides at various concentrations intersect one another at approximately 3300 c m ~ \ Mernagh and Wilde (1989) divided the spectrum into two regions. The skewing parameter is a function of two integrated areas: Xis equal to the integral between 2800 and 3300 cm~^, and

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Y is equal to the integral between 3300 and 3800 cm~^. The regression formula is given by wt%NaCl = a ^ f ^ ( 2 - ^ ) - ^ ,

(1)

where /? = 3(3400 c m " ^ 3 ( 3 2 0 0 cm"^) and a and j8 are experimentally determined regression parameters obtained from solutions of known concentrations for each spectrometer. The technique can be used up to halite saturation and can detect as Httle as 1 wt% NaCl in solution with a relative error of 15%. In the absence of other methods for the analysis of individual ions inside fluid inclusions, this technique is very useful, especially when the ice-melting temperature cannot be measured because of the presence of clathrates, or, in the case of complex systems, if phase diagrams are not available. Other solids, such as carbonates (Jrad et al., 1989), bicarbonates, phosphates and sulfates, are strong Raman scatters, and can be easily identified in fluid inclusions. An example of such solid identification in multiphase, fluid inclusions in a gold deposit is illustrated in Fig. 16 (Guilhaumou et al., 1990). Solids as small as 1 |xm in diameter can be identified. SiUcates such as K-feldspar, quartz and muscovite can also be easily determined (Coelho, 1990), unless they are iron-rich, in which case they absorb the exciting radiation and are often fluorescent. In contrast, NaCl and KCl have no first-order Raman spectra. Thus, these minerals cannot be identified by this technique. Carbon is often identified by its Raman spectrum by focusing the laser beam on the wall of the cavity, even though it is not visible under optical-microscopic examination. A very thin carbon coating around the wall of the cavity is thus indicated. As visible light is not significantly absorbed, the thickness is probably less than a few nm. 2. Hydrocarbon-fluid

Inclusions and Diagenetic Fluids

These inclusions are reUcs of the secondary hydrocarbon migration formed during the diagenesis of organic-matter-bearing sediments. They are also found in oil reservoirs, and could contain trapped hydrocarbons which are not necessarily identical to present-day oil; they thus can provide information on the time evolution of petroleum chemistry. There are very few studies in which micro-Raman spectroscopy has been found suitable for the characterization of hydrocarbon-fluid inclusions (Guilhaumou et al., 1981; Goffe, 1982; Pironon and Barres, 1990). The comparison of the Raman spectrum obtained from a natural fluid inclusion with that of a filled, synthetic inclusion permitted Pironon and Barres (1990) to identify the principal alkane present to be n-heptane. This identification was also consistent with micro-infrared data. Usually, the color of hydrocarbon inclusions is yellow

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Raman shift (cm-"') Figure 16 (a) Fluid inclusions with several solid phases as identified by micro-Raman spectra, (b) Raman spectra: siderite (sid), rutile (R), poorly oriented carbon (C) and quartz host crystal (Qz) (Guilhaumou et al., 1990).

to brown, indicating the absorption of light in the visible spectral range. They are, therefore, highly fluorescent under laser excitation with either the visible radiation provided by the Ar"^ laser (488 or 514.5 nm) or the Kr"^ laser (647.0 nm). The Raman spectrum is completely hidden by the highly fluorescent background, making analysis of the inclusion impossible with conventional techniques. The development of methods of treating fluorescence noise, or fluorescence rejection with the use of time-resolved, Raman spectroscopy, as described in the Introduction to this chapter, will be invaluable in future work.

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Inclusions can also be destroyed as a result of light absorption, provoking photochemical reactions and heating, and inducing chemical reactions such as hydrocarbon cracking. The experience of these authors has shown that these mixtures are fluorescent if they contain hydrocarbons heavier than CH4, even at low concentration, as indicated by a homogenization temperature of approximately -66°C. This result strongly suggests that the thermal stability of fluorescent organic molecules is roughly similar to the thermal stability of ethane, rendering micro-Raman spectroscopy useless for such hydrocarbon-fluid-inclusion analysis with visible excitation, at least with the use of conventional techniques. Fluids of the Terres Noires in the French southeastern Alps provide an exception to this empirical correlation. Guilhaumou et at. (1988) have correlated the regional distribution of wet and dry gases with vitrinite reflectance, and clay-mineral paragenesis. Methane, with a few mol% of ethane and perhaps propane, was identified in a region where the trapping temperature was in the 140-180°C range. Methane is the only hydrocarbon in the region where the temperature was between 180 and 230°C. Hydrocarbons heavier than methane are no longer stable at temperatures above 190°C, as shown from observations of fluid inclusions in oolitic limestones from core samples at 6.5 km depth (Guilhaumou et al., 1984). A notable exception to these generalities is the preservation of hydrocarbons to 300-320°C, as documented from fluid-inclusion analysis in high-pressure metasediments (Goffe, 1982). The attainment of high pressures due to nappe piling (up to 6 kbar), as inferred from mineral assemblage, strongly inhibited thermal cracking of the hydrocarbons. Fluorescence, and, more generally, light absorption by a material under monochromatic Hght illumination of energy EQ = hvQ, occurs if the energy difference between the electronic ground level and the excited electronic level of the illuminated material is similar in magnitude to, or smaller than, EQ. Therefore, an increase in the wavelength of the exciting radiation, resulting in a decrease of EQ, would be expected to eliminate the fluorescence. Thus, near-infrared excitation at 1.06 jjim provided by a continuous YAG laser has been used to study hydrocarbon inclusions (Pironon et aL, 1991). The FT Raman spectra were recorded with a Bruker IFS 66 spectrometer equipped with a Raman module FRA 106. The laser beam enters a classical optical microscope through an optical fiber. The fluid-inclusion sample was flat and jagged, but large in size (400 jxm); it was included in halite. It was filled with an aqueous phase with small crystals of anhydrite, a hquid-hydrocarbon phase with low fluorescence under UV iUumination, and a vapor bubble. The fluorescence on near-IR excitation was much lower than the fluorescence obtained under 514.5 nm excitation, but was still present. The Raman spectrum exhibited only the symmetric and antisymmetric —CH2 and —CH3 vibrations between 2800 and 3000 cm~^ and two small peaks of the C—H bending vibrations at 1300 and 1445 cm~^ (Fig. 17). The skeletal deformation vibrations in the 100-700 cm~^ region were not detected. The poor quality

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Raman shift (cnr'') Figure 17 Spectra obtained from a natural, multiphase-hydrocarbon fluid inclusion inside an NaCl crystal. FT-IR spectrum obtained with the use of a microscope coupled to the spectrometer; Raman spectrum obtained with 1.06 |xm excitation and an NIR-FT spectrometer coupled with a microscope. VIS-Raman: fluorescence spectrum obtained under 514.5 nm radiation (Pironon et al., 1991).

of the spectrum results from both the incomplete elimination of the fluorescence background and the decrease in the intensity of the Raman signal of frequency v-^ on decreasing the frequency of the incident radiation [see Chapter 1, Eq. (7)]. For instance, the intensity of a Raman signal with a wavenumber of 2900 cm~^ obtained with 1060 nm excitation is theoretically 41 times less intense than the intensity obtained with 514.5 nm excitation, for the same irradiance at the sample and the same efficiency of the optical system. In addition, the irradiance at the sample is at least four times weaker under 1064 nm radiation than with that at 514.5 nm. A priori, micro-infrared spectroscopy may prove more suitable than micro-Raman spectroscopy for the analysis of hydrocarbon-fluid inclusions, since a fluorescence signal does not interfere with the vibrational spectrum of the molecules. However, the host mineral of the inclusions, usually quartz or carbonate, strongly absorbs light below 2000 cm~^, rendering the micro-infrared technique of Uttle value

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for such studies. Although the Raman bands characteristic of the hydrocarbon skeleton are much weaker than the C—H stretching bands, the transparency of the host mineral gives a potential advantage to the micro-Raman over the micro-infrared technique. In future investigations of this type, it should be worthwhile to explore alternative methods for eliminating the fluorescence background from the Raman spectra.

3. Fluid Inclusions in the C—O—H—N—S System The success of micro-Raman spectroscopy in the Earth sciences is due to a large part to its application to the characterization of simple molecules in the C—O—H—N—S system. Those components which have been identified include: liquid and vapor H2O, CO2, CH4, N2, H2S, SO2, CO, COS, H2 and O2. The characteristic wavenumber values of the different diagnostic bands have been compiled by Schrotter and Klockner (1979). A typical Raman spectrum of CO2 and CH4 in a synthetic-fluid inclusion (Frantz et al., 1993) is shown in Fig. 18.

(a) Theory and practice of gas analysis The purpose of using micro-Raman spectroscopy in fluid inclusion analysis is to obtain reliable relative concentrations of the constituent gas-phase components. For this reason, the factors involved in the quantification of the analytical data and their caUbration are described below. For a low-density vapor phase irradiated by laser radiation of wavenumber VQ focused on the sample, the intensity of the scattered Raman line of wavenumber vij collected inside a small soHd angle (dfl), and measured by the spectrometer for a component /, is proportional to the integrated area. This quantity is given by Im(i, vo. Vj) = ^o(^o)'Nr-^^

(/, PQ, Vij, e) 'f(vo, Vj, pol),

(2)

where /o(^'o) is the irradiance at the sample provided by the laser radiation of wavenumber VQ, Ni is the number of moles of component / inside the irradiated volume and (do-/ y/dfl)(/, VQ, vij, 6) is the Raman differential scattering cross-section (RDSC) per mole of the Raman line of component / with wavenumber vij obtained by irradiation at wavenumber VQ. The significance of the angle d is described below. The factor fiy^, Vij, pol) represents the efficiency of the optical system, including the objective, the different lenses, the gratings and the detector, for radiation at absolute wavenumber PQ- P^J with a given polarization (pol). Raman differential scattering cross-sections have been tabulated by

330

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Figure 18 Raman spectra of carbon dioxide (a) and methane (b) contained in a synthetic fluid inclusion (Zhang and Frantz, 1992).

Schrotter and Klockner (1979) for many gas-phase species of interest. However, these RDSC values were measured at low vapor densities with the use of a different geometry from that of the typical (nominal 180'') scattering geometry employed in the Raman microprobe. In addition, the refractive index of the host mineral (>1.5) of the inclusion produces a complicated optical path, and the birefringence of the mineral and any resulting optical

Earth, Planetary and Environmental (a)

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331

OBJECTIVE

P-BOpm N h - 1 N m - 1 . 5 5

Figure 19 Optical paths of light focused inside a transparent mineral with the use of (a) an immersion objective or (b) a dry objective.

rotation, as is the case for quartz along the c axis, lead to complications which must be considered (Bremard et al, 1987). The laser power at the output of the objective can be measured, but the irradiance at the sample /o(^'o) depends strongly on the optical path from the surface of the sample to the inclusion itself. The laser beam crosses at least two interfaces: air-mineral and mineral-fluid inclusion. Light reflection and refraction occur at all such interfaces, and cannot be quantified in the present case, because the exact shape of the inclusion is unknown. Therefore, /o(^o) cannot be evaluated and an absolute intensity measurement is impossible. However, relative determinations of the species inside a given inclusion can be made. The mole fraction {Xi = Ni/l^Ni) can be measured with the use of Eq. (2), provided that the RDSC and/factors are known. The RDSC of gaseous N2 (Q branch) has been accurately measured, and wavelength-normalized relative values (2) for other gas-phase species have been tabulated (Schrotter and Klockner, 1979). Usable relative RDSC values (a) are given by Dubessy et at. (1989). Dry objectives can focus the laser radiation in air or vacuum into a beam with a minimum diameter of approximately 0.5 fxm. This ideal situation no longer holds if the laser is focused inside a transparent material, due to refraction at the air-mineral and mineral-inclusion interfaces (Fig. 19). In addition, the incident beam is attenuated as it traverses the sample, decreasing the irradiance at the inclusion. These factors make it difficult to analyze fluid inclusions of 5-20 iJim diameter, situated at depths greater than 80 |xm within the host mineral. The loss in intensity due to refraction can be minimized with the use of immersion objectives, commonly with oil or

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water as the immersion medium. Because oil is an alkane, CH4 cannot be detected with this immersion medium as the intense C—H stretching of the oil overlaps the symmetric stretching vibration of CH4, even when a spatial filter or confocal-hole optics is used. Water has a smaller refractive index than oil, but can give good results: a five-fold increase of the Raman signal has been obtained with the use of a water immersion objective (Dubessy et at., 1986). The efficiency of the optical system, f{vQ, ^/y, pol), depends both on the wavenumber {VQ— vi j) of radiation and the polarization of the light. The polarization state of the scattered radiation at the entrance sUt of the spectrometer is questionable in micro-Raman experiments. First, the use of high numerical aperture objectives results in a sUght depolarization of the laser radiation, and the beamsplitter used to separate the incident and scattered radiation also depolarizes the scattered radiation (see Chapter 2). In addition, the host mineral itself, if birefringent, can affect the polarization state of the scattered radiation (Turrell, 1989). For low vapor density, the depolarization ratio of the Raman lines of the different gas components is zero by symmetry {vi of N2 and CH4) or experimentally very small for CO and CO2. Thus, the polarization state of all of the Raman lines scattered by the vapor phase can be considered to be identical in a first approximation. A polarizer situated just before the entrance sHt of the spectrometer, and oriented perpendicularly to the grating grooves, permits a comparison to be made of the intensity of the Raman scattered radiation emitted by the different components. The polarization of the Raman scattered light before the polarizer varies from one inclusion to another, depending on the depth of the inclusion and the crystallographic orientation of the host mineral. Therefore, an optimization of the scattered Raman intensity can be obtained by rotating the inclusion around the optical axis of the microscope. The spectrometer function, including the wavelengthdependent response of the polarizer, the spectrometer and the detector, is measured with the use of an intensity-calibrated, standard white lamp. The differential scattering Raman cross-section contains a geometrical term which is a function of the angle 6 between the direction of observation and the polarization vector of the linearly polarized excitation (Placzek, 1934; Schrotter and Klockner, 1979), namely, d^U/

r,.

.

(^

^ . (l-pjsin^^

.^.

where p^^^ is the depolarization ratio of the Raman band at wavenumber If the laser beam is focused through a high-aperture objective directly into a low-density vapor phase with a low refractive index (w« 1), the incident elemental laser beam rays at the sample include a solid angle ft relative to

Earth, Planetary and Environmental

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the optical axis of the microscope. In addition, the Raman-scattered light is collected over all directions inside the solid angle of the objective. A complete calculation takes into account the incident energy distribution across the laser beam after the objective. This analysis yields a quite complex 6 distribution of the incident and scattered intensity, with the result that the scattering geometry is expected to have an influence on the Raman scattering cross-sections (Bremard et al., 1985, 1986, 1987; Turrell, 1985). However, a simpHfied integration over 6, taking into account the sUghtly different depolarization ratios for the different compounds, but ignoring the 6 dependence of the incident intensity, introduces only a very small departure from the relative Raman scattering cross-sections experimentally measured with the use of the 90° scattering geometry. In the case of fluid inclusions, the laser is focused through a host mineral and is refracted. First, consider the case of a nonbirefringent mineral. The refraction produced by the host mineral, as weU as the spatial filter or confocal pinhole used to enhance the spatial resolution along the optical axis, implies that the scattering volume actually seen by the spectrometer is irradiated principally by the elemental rays which make small angles with respect to the optical axis of the microscope. These rays are also the most intense for a laser working in a TEMQO mode (see Chapter 3 and Fig. 19). Consequently, the geometry of the scattering from a fluid inclusion cannot significantly modify the Raman scattering cross-sections which are measured in the classical scattering geometry with a macroscopic sample for which 6 = 90°. The presence of birefringence would not be expected to introduce any additional effects, since the same argument can be made for each component. The use of low-pressure, standard gas mixtures in a microscopic cell such as that designed by Chou et al. (1990), provides an easy and rapid method to derive the instrumental efficiency coefficients in an optical configuration identical to that used during fluid inclusion analysis (Wopenka and Pasteris, 1987). An additional problem concerns the appUcability to fluid inclusion analysis of the tabulated. Raman scattering cross-sections, which were measured at low vapor density Furthermore, Placzek's polarizability theory of freely rotating molecules, is not generally appropriate to fluids with strong intermolecular interactions. To address these questions, the dependence of Raman scattering cross-sections on composition and density must be considered. The first source of modification of the Raman differential scattering cross-section is the internal field effect, which can be represented by (dcr/dfl)* = L(da/dfl). The correction factor L is given by Onsager's model (Schrotter and Klockner, 1979) in the form

334

P. F. McMillan et al.

where UQ is the refractive index of the medium at the wavelength of the exciting radiation and n^ is the refractive index of the scattered radiation. For a pure CO2 fluid at 106.72 atm pressure and 25°C, corresponding to a density of 1.23 gcm~^ (Angus et al., 1976), the refractive index variations are - 8 x 1 0 " ^ per 1000 cm~^ in the 4922-5016 A and - 5 x 10"^ per 1000 cm" 1 in the 5016-5876 A spectral range (Michels and Hamers, 1937). In the calculation of the mole fraction Xi, the refractive indices of all of the Raman lines can be considered to be equal, eliminating the internal-field effect for the RDSC relative to N2. This approximation assumes that the internal-field effect simply enhances the Raman intensity by the same factor for all of the gas components (Dubessy et al., 1989). However, careful intensity measurements on pure N2 have recently shown that the internal-field effect cannot account for all of the increase in the Raman scattering cross-section at high densities (Fabre et al., 1989). A correction term if/ is introduced to account for this additional increase in the Raman intensity, such that, (T* = (TLII/. It is important to determine any variation in i// as a function of composition, pressure or density. At the present time, several contradictory observations have been reported. First, a natural fluid inclusion of the CO2-N2 system presented a critical homogenization at 27°C, which fixed the composition at 5 mol% N2. The composition calculated with a one-bar relative-Raman scattering cross-section yielded the same value (Diamond, 1986; Dubessy et al., 1989). This result suggests at least that the variation of if/ with density is not significant or that it is the same for the two gas components at this composition and moderate density. The Raman spectrum of CO2 in the symmetric stretching region is characterized by a Fermi resonance between vi and 2i^2 which exhibits two components. In a pure CO2 fluid the intensity ratio of the two components of the Fermi diad is a function of density (Garrabos et al., 1980; Van den Kerkhof, 1988a), although in principle the total intensity of the diad remains constant. Therefore, it is the relative Raman intensity of the two components of the diad that must be measured as a function of density. On the other hand, Pasteris et al. (1990) and Chou et al. (1990) measured the ratios of the areas of the vi bands of CH4 and N2 in a 90mol% N2-IO mol% CH4 and a 50 mol% N2-50 mol% CH4. These mixtures were investigated at pressures from 6.9 bar to more than 600 bar. These authors found a significant variation up to 75 bar, with no further variation above this pressure (Fig. 20). Measurements carried out on a 45 mol% N2-55 mol% CH4 mixture from 10 bar to 2.5 kbar show that the area ratio of the two bands remains constant over the entire pressure range (Fabre and Oksengorn, 1992). The disagreement between the two sets of data indicates clearly the need for more experiments to resolve this problem. These data should be obtained with both the classical, macro-spectroscopic cell, and the microspectroscopic cell, such as that described by Chou et al. (1990). The presence of intermolecular forces also produces other effects in the

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Raman spectra, affecting the frequency shifts, the linewidths and the bandshapes (Srivastava and Zaidi, 1979). The measured lineshape is a convolution of the natural Raman lineshape and the instrumental response function. Experimental measurements and calculations (Wang, 1987) have shown that a spectral resolution smaller than one-fifth of the width of a band is required to avoid increasing the width, and thus modifying its shape. This condition is only achieved with a spectral resolution better than 1 cm~^ for the gases of interest; it thus requires a narrow slit, resulting in insufficient signal intensity in the spectra of most of the inclusions investigated. The background of the signal is not always fiat, which is not favorable for bandshape analysis, a technique which requires perfect scattering conditions. In contrast, the line shift is much easier to measure under the experimental conditions used for fluid inclusions, as was first documented by Dhamelincourt et at. (1979) in his investigation of natural, CH4-bearing fluid inclusions. The internal pressure in the inclusion can be measured from the observed frequency shifts provided that the pressure shift has been previously calibrated. This measurement has been made by Chou et at. (1990) up to 600 bar at room temperature. Pure, CH4-bearing fluids are rather rare and phase transitions observed by microthermometric studies are sufficient to determine the pressure, and thus their densities. However, more interesting data concern gas mixtures, for which the observed, phase-transition temperatures are not always easily interpretable. CaUbrations of the shift of the vi Raman band of CH4 have been made for two compositions in the CH4-N2 system, as shown in Fig. 20 (Chou et al, 1990; Pasteris et a/., 1990). The shift with pressure of the N2 lines, as well as those of the Fermi resonance

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diads of CO2, are much smaller, and the accuracy in the pressure determination from these shifts is expected to be insufficient for the interpretation of the spectra of fluid inclusion. Detailed calibrations are also necessary in this domain.

(b) Water analysis A fluid inclusion usually traps a single fluid phase under the P-T conditions of fluid circulation. In contrast, at room temperature, a fluid inclusion is usually an association of an aqueous fluid phase with dissolved gases and ions, and a volatile phase with a Hquid or vapor which contains the simple gases such as described above. However, in fluid geochemistry the bulk V-X properties of the included fluid must be known. These quantities are the only variables which are representative of the P-T conditions of fluid circulation. Under heating, the two fluid phases homogenize into a single fluid. Because the composition and the molar volume of each phase are generally quite well known, and to a first approximation, fluid inclusions are constant volume systems, the determination of the bulk composition alone is sufficient to derive the bulk molar volume. A priori, the bulk compositional analysis could be made when the inclusion is heated above its homogenization temperature, at which point it forms a single fluid phase. The condition for obtaining reUable analytical data of this type is the knowledge of the Raman scattering cross-section of each component for a wide range of temperatures, fluid compositions, and densities. The interpretation of the Raman spectroscopy of water is a matter of intense debate, because the bands in the O—H stretching region are affected by both inter- and intramolecular interactions (Hare and Sorensen, 1992). Very few Raman data are available on pure water as a function of pressure and temperature (Lindner, 1970; Ratcliffe and Irish, 1982; Kohl etal, 1991; Frantz et al., 1993). Intermolecular coupling is due to hydrogen bonding and dipole-dipole interactions, and thus is very sensitive to the local environments of the water molecules. These interactions depend on the temperature and pressure, as well as the nature of neighboring species. The latter can include other water molecules, nonpolar or dipolar neutral molecules, or ions. For instance, the influence of ions on the O—H stretching vibration of water at room temperature has been documented (Walrafen, 1964, 1966, 1967; Rull and de Saja, 1986; Walrafen et al, 1988). No systematic study has been carried out on water-gas mixtures. However, prehminary data (Fig. 21) on a water-poor, natural fluid inclusion and two synthetic fluid inclusions of the H2O-CO2 system show important variations in the peak positions and FWHM values of the O—H stretching Raman bands (Dubessy et al., 1992a). Therefore, it is not possible at present to use the Raman spectra of fluid

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CO

z LU

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3700

3600

3500

3400

1500

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Raman shift (cm ^ ) Figure 21 Raman spectra of the stretching vibrations of water and of the Fermi resonance of carbon dioxide contained in fluid inclusions of different compositions, (a) r = 200°C, no visible water; (b) 7 = 300°C, X(C02) = 0.5, X(H20) = 0.5; (c) r = 300°C, X(C02) = 0.3, X(H20) = 0.7 (Dubessy et aL, 1992).

inclusions, as obtained from the single-fluid phase above the homogenization temperature, to determine the bulk composition. It will be necessary to carry out extensive experimental and theoretical studies on the Raman spectroscopy of gases and water as functions of composition, density and salt concentration to resolve this problem. (c) Coupling of microthermometry

and micro-Raman

spectrometry

Gas components are involved in phase transitions such asS + L + V - » L + V or S -h L (or V) —> L (or V) for which the temperature is measured by microthermometry. Phase diagrams can be used to interpret the temperature

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P. F. McMillan et al.

of phase transitions only if the solid which disappears is identified. The soUd phase can easily be determined at a given temperature with the use of a microthermometric stage, as for salt hydrates. For instance, it has been possible to discriminate between the melting of sohd H2S and solid CO2 in two distinct inclusions in which melting of an unknown solid phase was measured at approximately -100°C. Upon cooling, a gas-water fluid inclusion nucleates clathrate phases. In saline inclusions, optical observation alone is not sufficient to decide if the solids are clathrates or salt hydrates. The Raman spectra of clathrates are composed of two kinds of band: O—H stretching bands which are similar to the spectrum of hexagonal ice, and vibrations assigned to the gas molecules located in the cages. The Raman spectrum of a CO2-CH4 clathrate, investigated in a synthetic fluid inclusion, consisted of bands at 2903.5 and 2913 cm~^ for the CH4 molecule, whereas only a single high-wavenumber diad component for CO2 was observed. It was only slightly shifted to 1380cm~^ (Seitz et al., 1987). This observation led the author to suggest that CO2 occupies only one cage, whereas CH4 occupies two. In addition, intensity measurements of the CO2-CH4 fluid phase in the presence of clathrates, and after the dissolution of clathrates, indicated that CH4 preferentially partitions into the clathrate relative to the carbonic phase. Inverse partitioning has been found in other fluid inclusions (Seitz and Pasteris, 1990), indicating that the partitioning is a complex function of temperature, pressure and composition. Careful studies of the topology of the phase equilibrium in the CO2-CH4N2 system, based on the combined use of microthermometry and microRaman spectrometry, have been carried out by Van den Kerkhof (1988a, b, 1990). This type of work provides useful constraints for fluid inclusion studies, and for theoretical modelling of phase equilibria. In general, the determination of the V-X properties of the aqueous and nonaqueous phases in fluid inclusions is carried out through the combined use of microthermometry, micro-Raman spectrometry, and computer modeling of appropriate phase equilibria (Van den Kerkhof, 1988a,b, 1990; Dubessy et a/., 1992b). In this sense the use of micro-Raman spectrometry for the analysis of molecular species in fluid inclusions has strongly stimulated the modelling of the phase equilibria involved in these systems. These results augment the information obtained from the Raman spectra.

(d) Geochemical applications of the determination of the gas content (i) Redox states of paleo-fluids and the metallogenetic consequences. The composition and density of fluid inclusions of the C02-CH4-H20-H2S-NaCl system can be calculated by combining microthermometric and micro-Raman data. These data can be interpreted from the relevant phase equilibria. Assuming chemical equilibrium among the molecular species, and with the

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use of an appropriate equation of state to calculate the fugacity coefficients of each species and the fluid pressure at each temperature, it is possible to estimate the oxygen fugacity (/02) ^^^ the sulfur fugacity (/S2) from the following chemical equilibria (Ramboz et al., 1985; Dubessy et al., 1989): CH4 + 2 0 2 ^ - ^ C 0 2 + 2H20

(5)

CH4 + 2H2O + 2S2 ^ - ^ CO2 + 4H2S.

(6)

For example, fluids found in quartz veins often do not contain any associated minerals, although they might provide information on the redox state of the fluid. However, this result can be obtained for the fluid inclusion itself from the above equilibrium expressions. It has also been shown that the temperature of trapping of C02-CH4-H20-H2S-bearing fluids can be deduced from the fluid-pyrrhotite equilibrium, if the fluid inclusions are cogenetic with pyrrhotite (Dubessy et al., 1990). In this case the V-X properties of the fluid inclusions provide one relationship to the temperature [/s2 ~ ^(^)]y while the pyrrhotite composition yields another [/s2 = ^ ( ^ ) ] This result allows the temperature to be determined by simultaneous solution of this system of two equations. The temperature calculated by Dubessy et al. (1990) has been found to be consistent with independent determinations from rock-mineral assemblages. Furthermore, if graphite is present in the surrounding rocks, chemical equilibrium between the fluid and graphite can be checked by substituting the fco2 ^^^ /02 values calculated at the trapping temperature and pressure of the fluid inclusions into the mass-action law of the heterogeneous equilibrium: C + 0 2 ^ ^ C 0 2 . In one occurrence of a quartz-wolframite vein in graphite-bearing metapelites (Ramboz et al., 1985), it was shown that the composition of the fluid inclusions implies the attainment of fluid-graphite equilibrium down to 420°C. This blocking temperature in the C—O—H system is of paramount importance, since it controls several major parameters, such as the density gradient of fluids circulating around magmatic bodies, intruding graphitic series, and the redox state of the fluid. Uranium, tin and tungsten sites often overlap in the western European hercynian belt. However, uranium deposits are never associated with Sn-W mineralizations. Since these deposits exhibit a similar behavior at the magmatic stage, it is probably the hydro thermal stage which controls their spatial distribution. Dubessy et al. (1987) used micro-Raman spectrometry of fluid inclusions associated with these metal deposits to demonstrate that the redox state of the fluid was the key parameter. The/02 of fluids associated with tin and tungsten deposits has been shown to he between the Ni-NiO and quartz-fayalite-magnitite (Q-F-M) oxygen buffers for temperatures of fluid circulation between 550 and 400°C. These ore deposits often occur in environments which contain graphite. Solubility measurements of cassiterite (Sn02) have shown that fluids with a redox state close to that defined by

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the Q-F-M buffer values favor the transport of tin, a prerequisite for further efficient deposition. Hematite is a frequent gangue mineral of uranium deposits, and its presence corresponds to fo^ values at least four orders of magnitude higher than those for fluids associated with tin and tungsten deposits. No CH4 has been found in fluid inclusions associated with uranium deposits formed around 400°C. Solubility data for UO2 show that high-/o2 values favor uranium transport in the fluid. Fluids percolating through graphite-bearing sediments at temperatures above 400°C can never achieve /02 values near those of the hematite-magnetite buffer. Therefore, the fluid composition, and the resulting values offo^, provide the key parameter which accounts for the difference in the behavior between uranium and tin-tungsten at the hydrothermal stage. In addition, this analysis explains the scarcity of uranium deposits formed between 400 and 500°C. Carbon-bearing sediments are common, thus fixing fluid-phase fo^ at low values. The extraction of uranium from source rocks is inefficient at these values and therefore so is its deposition. Fluids associated with gold deposits have been also extensively documented. These systems generally show /02 values near those of the Ni-NiO buffer (Charoy and Gonzalez-Partida, 1984; Boiron etaL, 1988, 1990; Touray et al., 1989; Wu et al., 1989; Guilhaumou et al., 1990; Ortega et al, 1991). (ii) Nitrogen. Molecular nitrogen is the main component of the Earth's atmosphere, but Httle was known about this species in geological fluids before the advent of fluid-inclusion analysis by micro-Raman spectrometry. N2C02-bearing fluids have been identified in inclusions in quartz and dolomite from a triassic-salt diapyr in Tunisia (Guilhaumou et al., 1981). The N2 mole fraction in these inclusions was variable, and attained a value of 0.92. A nonfluorescent, aromatic hydrocarbon was also found in some of these inclusions, indicating an organic origin for the nitrogen. N2-rich fluids have also been documented in many metamorphic and geothermal environments (Bastoul, 1983; Touret and Dietvorst, 1983; Bussink et al., 1984; Cheilletz, 1984; Giuliani, 1984; Wilkins and Dubessy, 1984; Casquet, 1986; Darimont, 1986; Bottrell et al., 1988; Darimont et al., 1988; de Alvarenga et al, 1990; Cathelineau et al, 1990; Wilkinson, 1990). Organic matter, or the products of its thermal maturation, is present in all of these environments. Ammonium, of proteinic origin, can be stored in clay minerals, provided that early diagenesis occurs in anoxic conditions (Dubessy and Ramboz, 1986) and it is incorporated into micas and feldspars (Hallam and Eugster, 1976; Honma and Itihara, 1981; Duit et al, 1986). Devolatilization reactions or K~^/NH4' exchange reactions release NH4^ into the fluids, where it is subsequently transformed into H2 and N2, the most thermodynamically stable nitrogen-bearing species. Therefore, organic matter may constrain deep geochemical processes, not only through C and S elements, but also through

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N, as indicated by the N2-bearing fluid inclusions and the presence of ammonium-bearing minerals. The identification of N2-rich fluids has stimulated a new field in geochemistry which now needs additional experimental data for modelling fluid-mineral equilibria in nitrogen-bearing systems. (iii) Sulfur. As noted earlier, sulfate species (with sulfur oxidation number +6) are quite rare in fluids from the earth's crust, because the redox state of these fluids, controlled by rock mineral assemblages, is too low. In contrast, H2S has often been found in fluid inclusions representing diagenetic fluids (Dhamelincourt et «/., 1979; Beny et aL, 1982; Guilhaumou et al., 1984), and in hydrothermal fluids (Boiron et al, 1990; Dubessy et al, 1990). However, the abundance of iron and other metal elements in the rocks favors the growth of sulfide minerals and thus often fixes the H2S content of hydrothermal and metamorphic fluids at low levels - below the detection limit for micro-Raman spectrometry. This limitation is not present for the case of evaporite rocks metamorphized by the implantation of dolerite siUs in Siberia (Grishina et aL, 1992). Concentrations of H2S up to 30mol% were documented in some inclusions. Amorphous or crystalline sulfur was also identified, coexisting with a dense, C02-rich phase containing H2S ( < 6 m o l % ) . Hydrogen sulfide and Sg probably originate from the reduction of sulfate. In addition to these usual sulfur compounds, COS was identified for the first time in individual fluid inclusions at a concentration between 0.5 and 1 mol%, with the use of both micro-infrared and micro-Raman techniques. The COS formation is probably related to the equihbrium of the reaction: CO2 + H 2 S ^ C O S + H2O (Grishina et al, 1992). In this study, the CO2/S8 ratio was found to vary from one inclusion to another. This series of fluid inclusions can be used as an experimental cell to monitor the evolution of sulfur speciation with temperature. Upon heating, amorphous or orthorhombic Sg melts and dissolves in the C02-rich fluid phase. No Raman signal from any polysulfide species was found above 150°C, suggesting that all sulfur was monatomic. Therefore, a fascinating sulfur chemistry seems to exist in such special environments, as documented by fluid-inclusion studies. These results call for further experimental work to achieve a better understanding of these systems. (iv) H2 diffusion: origin and re-equilibration of fluid inclusions. An important question in the use of fluid inclusions for the study of geochemical fluids is to know if the content of the inclusion is likely to have been modified after trapping, by diffusion under a chemical gradient between the trapped fluid and an external circulating fluid. Molecular hydrogen has always been the best candidate for altering the initial composition, and has often been proposed to explain unexpected fluid compositions. It has been identified in fluid inclusions from several environments. In the uranium deposit of Rabbit Lake, H2 coexists with O2 inside the same vapor bubble (Fig. 22).

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H2

Em

Ea

1520

1580

UJlw

4130

4190

Raman Shift (crrr'^) Figure 22 Identification by micro-Raman spectroscopy of coexisting molecular oxygen and hydrogen in the vapor phase of a fluid inclusion from the Rabbit Lake uranium deposit in Saskatchewan, Canada (Dubessy et al., 1988).

This unusual composition, in complete chemical disequilibrium, appears to result from the radiolysis of water in contact with uranium ore before fluid trapping (Dubessy et al., 1988). Pure H2 fluid inclusions or H20-H2-bearing inclusions with less than 0.5 mol% CH4, were found in the nuclear reactor zone of the famous Oklo uranium deposit (Gabon), where chain fission reactions occurred between 1.5 and 1.7 bilHon years ago (Dubessy et al., 1988). Molecular hydrogen was produced by the radiolysis of water in the presence of organic matter by the intense nuclear radiations when chain fission reactions took place at between 100 and 200°C. H2 was also identified inside inclusions from the Illimaussacq alkaline intrusion (Greenland), coexisting with methane and ethane (Konnerup-Madsen et al., 1985). Finally, H20-H2-bearing inclusions were documented in the aureole growth of a diopside from the Malenco peridotite in the Swiss Alps (Peretti et al., 1992). These fluid inclusions were formed at around 400°C during the alpine metamorphism which affected the serpentinite; they were contemporaneous with opaque minerals such as heazlewoodite (Ni3S2), awaruite (NisFe) and magnetite (Fe304), indicating high H2 fugacity. The concentration of H2 in the inclusions is consistent with the redox state calculated from the mineral assemblage. All of these case studies indicate that H2 can be relatively well preserved inside natural fluid inclusions, at least for temperatures below

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400°C. In contrast, fluid inclusions of the massive sulfide deposits (Ducktown, Tennessee, USA) have a CH4/CO2 ratio much higher than the value calculated from the redox state of the fluid derived from the rock mineralogy (Hall et at., 1991). These authors concluded that the present-day fluid composition results from an introduction of molecular H2 from the ambient fluid at 450°C. The introduction of hydrogen in synthetic-fluid inclusions has been investigated experimentally at 718-728°C (Morgan et al, 1993), 650°C (Hall et aL, 1989) and 600°C (Morgan et aL, 1993; Plyasunova et al., 1993). The comparison of these results with those obtained on natural samples clearly shows that both the gradient in H2 fugacity and the temperature control the efficiency of H2 diffusion through quartz. Similar experiments have been carried out on initially pure C02-fluid inclusions contained in olivine (Pasteris and Wanamaker, 1988). The samples were heat-treated at atmospheric pressure in a gas-mixing furnace up to 1400°C. The redox state of the fluid was controlled by mixing appropriate volumes of H2 and CO, or CO and CO2. After this operation, CO was identified inside the fluid phase; inclusions treated at the lowest value of/02 contained moderately well-crystallized graphite. According to Pasteris and Wanamaker (1988), the most likely mechanism of re-equilibration involves the diffusion of metal vacancies within the host olivine. This work demonstrates the power of micro-Raman spectroscopy as a technique to monitor precisely the modifications experienced by fluid inclusions under chemical gradients. The determination of fluid speciation and kinetics of reaction in the C - O - H system are of paramount importance in fluid geochemistry. The thermal dissociation of oxalic acid dihydrate contained inside a sealed glass capsule was studied with the use of micro-Raman spectrometry to monitor the gas composition at room temperature (Morgan and Chou, 1992). The progress of the reaction CO + H2O ^^ CO2 + H2 and the loss of H2 with increasing temperature were determined by mass-balance calculations.

B. Glasses and Melts

Aluminosilicate-melt phases are involved in all current magmatic processes. Thus, there is much interest in their structure and dynamics, as well as in the glasses formed from them. Raman spectroscopy has been used extensively in structural studies of natural and synthetic aluminosiUcate glasses and melts to gain valuable insights into the molecular species present and the relationships between structure and bulk properties (McMillan, 1984a, 1989; Mysen, 1988). Micro-Raman spectroscopy has played a major role in this work. The simplest apphcation of micro-Raman techniques is to compositions which do not vitrify easily, which is especially true for metal-rich or peraluminous compositions, or for glass samples prepared from high

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pressures, or with high volatile content (McMillan and Piriou, 1982; McMillan et al., 1982; WilHams et at., 1989b; Xue et al, 1991). In these cases micro-Raman spectroscopy permits the study of luim-sized regions of glass, often in an intimate mixture of glassy and crystalline materials. In addition, micro-Raman spectroscopy is a technique of choice for studying the structural nature of silicate glasses and liquids at high pressures and temperatures.

1. Compositional Studies of Glasses Most Raman investigations of silicate and aluminosilicate glasses have been concerned with the changes in the spectra as the glass composition is varied systematically along a compositional join (Brawer and White, 1975; Mysen et al, 1980, 1982; Furukawa et al, 1981; McMillan et al, 1982; Seifert et al, 1982; Matson et al, 1983, 1986). This type of study has had some success in rationahzing observed systematic trends in the bulk physical properties of the glasses, or their corresponding liquids (Mysen et al, 1980; Navrotsky et al, 1982; Mysen, 1988, 1990). Considerable attention has been paid to the Raman spectrum of vitreous Si02 (Fig. 23a). This spectrum is characteristic of a disordered, fully polymerized network of corner-sharing Si04 tetrahedra (McMillan, 1984a, 1988). Within the series of alkali and alkaline-earth silicate glasses, strong, polarized Raman bands appear successively near 1100cm~^, 1000 cm~^, 900 cm~^ and 850 cm~^ with increasing metal oxide content, as shown in Figs 23b and c (Brawer and White, 1975; Mysen et al, 1980, 1982; Furukawa et al, 1981; Matson etal, 1983; McMillan, 1984a,b, 1988; Mysen, 1988, 1990). These bands have been assigned to the symmetric Si—O stretching vibrations of Si04 groups with 1, 2, 3 and 4 nonbridging oxygens (Q^, Q^, Q^ and Q^ species), respectively. The appearance of these bands is consistent with progressive depolymerization of the Si02 network on addition of metal oxide, hence the term 'network modifying cation' for the alkali or alkaline-earth metal. The relative intensities of these characteristic bands can be used to estimate the proportions of the different silicate-polymer species at a given composition, and hence the equilibrium constant for the speciation reaction (Mysen, 1988, 1990; McMillan et al, 1992b; Mysen and Frantz, 1992). The effect of compositional changes on the Raman spectra of the series of aluminosihcate glasses is less well understood (McMillan et al, 1982; Seifert et al, 1982; Matson et al, 1986). In general, the bands are much broader than those of the binary silicates, so it is already more difficult to identify individual components (Fig. 24). The broad, high-frequency region contains several unresolved bands due to the (Si, Al)—O stretching vibrations. The low-frequency region, between 400 and 700 cm~^, can be assigned to bending vibrations of the TOT (T = Si, Al) linkages.

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1000 500 Raman Shift (cm-"')

>LU

Na20 2*45 SiOe

Z

< u

CO

NaeO 1-75 Si02

< 0^

Na20 V5 Si02

Na20 1-22 Si02 ^ '

•

1200

Raman

" ^

800

400

Shift

0

(cm-'')

1000

Raman

500

Shift

0

(cm""')

Figure 23 (a) Polarized Raman spectra (parallel) of an Si02 glass (McMillan et al., 1982). (b) Polarized Raman spectra (parallel) of Si02-Na20 glasses (Furukawa et al., 1981). (c) Polarized Raman spectra (parallel) of Si02-CaMgSi04 glasses (McMillan and Piriou, 1983; McMillan, 1984b).

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300

600 900 Wavenumbers ^cm"')

1200

Figure 24 Raman spectra of an Si02 glass as a function of pressure, including the spectrum of the sample quenched from —30 GPa. Above 40 GPa the spectra weaken and no peaks were detected (Hemley et al., 1986a).

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2. Structural Studies of Silicate Glasses at High Pressures As noted earlier, the micro-Raman technique is uniquely suited for obtaining vibrational spectra of samples in situ at high pressures with the diamond-anvil cell. This technique permits a detailed study to be made of the response of the glass structure to applied stress, yielding an understanding of the atomic level rearrangements in the glass structure with increasing pressure. Hemley et al. (1986a, 1987a) used micro-Raman spectroscopy to observe the effect of pressure on the Si02 glass framework at room temperature (Fig. 24). In this work they found that the principal Raman band at 430 cm"^, which is due to the symmetric bending vibration of the intertetrahedral SiOSi hnkages, sharpened and moved to higher frequency with increasing pressure. This result indicates that the major compression mechanism involved is a closing of the SiOSi angles in the structure, at least at pressures up to approximately 20 GPa. At higher pressures, the Raman spectrum broadened and weakened significantly, which was interpreted by Hemley et al. (1986a, 1987a) as indicative of a coordination change about the Si atoms. Wolf et al. (1990b) carried out in situ high-pressure micro-Raman studies of Na2Si409 glass to 49 GPa in the diamond-anvil cell (Fig. 25). In this work, the high-frequency peak of Na2Si409 glass at 1100 cm~^, which is due to the symmetric stretching vibration of Si04 tetrahedra with one non-bridging oxygen (Q^ species), disappeared at pressures near 18 GPa. Previous ^^Si NMR studies on glasses quenched from up to 12 GPa showed the presence of large quantities of V- and Vl-coordinated species in high-pressure Na2Si409 and K2Si409 glasses (Stebbins and McMillan, 1989; Xue et al., 1989). The in situ micro-Raman results indicated that these high-coordinate silicate species were formed at the expense of Q^ units in the glass, most probably by attack of the nonbridging oxygen on an adjacent tetrahedral sihcon atom. It now appears that this modification not only provides a compression mechanism for high-silica siUcate glasses and their corresponding Hquids, but also a mechanism for oxygen-ion diffusion and viscous flow in the high-temperature hquids (Poe et al., 1992; Stebbins et al., 1992). At pressures above 20 GPa, Wolf et al. (1990b) observed significant broadening in the Raman spectrum of Na2Si409 glass (Fig. 25). This result was interpreted as an indication of the onset of a coordination increase around the bridging oxygen atoms, concurrent with a coordination change about the remaining silicons. These observations are analogous to those of Hemley (1987) and Hemley et al (1986a) on Si02. Kubicki et al. (1992) carried out a similar in situ high-pressure, micro-Raman and micro-IR study of glasses in the (Ca,Mg)Si03 system, to pressures of 40-50 GPa. Significant changes in the spectra were observed, which were again interpreted in terms of changes in the Si coordination number, consistent with the results of molecular dynamic simulations of high-pressure liquids and glasses (Angell et al, 1983; Kubicki and Lasaga, 1991; Rustad et al, 1991; Kubicki et al,

348

P. F. McMillan et al. COMPRESSION

49.0 GPa 42.0 GPa 37.0 GPa 32.8 GPa 28.3 GPa 23.0 GPa 17.8 GPa 13.1 GPa 8.8 GPa 4.9 GPa 2.5 GPa 1.0 GPa

1 atm

400 800 1200 Raman Shift (cm"^) COMPRESSION

MECHANISMS Q3

+ Q'*

.0^

"si'^^I^^^Si" /

^

\

/

\

^Si /

Si

SI //

SI \

/

\

/

\

Si 2Q3

+ Q'^

2Q^* + ^Sl

"Sl^ 0

0^Si

SI

/

^

SI

\

/

\

,Si /

Si ^

Si

Si

SI Q3 + Q4

^Sl Si " I ^ \ 0 0^Sl^

'Si I

0

^sK y

\

0

\

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1992). Sykes et al. (1993a) examined the micro-Raman spectra of KAlSiaOg and NaAlSi308 glasses quenched from pressures up to 10 GPa, but in this case no obvious evidence of coordination change was observed. This result is consistent with the NMR results (Stebbins and Sykes, 1990). The results of the in situ Raman spectroscopic work summarized above suggest that many of the major changes in the spectra of glasses as a function of pressure may not be quenchable. However, a number of Raman investigations have been carried out on glasses which were quenched from high-temperature melts prepared under high pressure (Sharma et al., 1919\ Mysen etal., 1983; McMillan, 1984a; Xue etal, 1991). These studies do show significant differences between the spectra of the densified glasses and those at atmospheric pressure, indicating that some of the equilibrated highpressure Hquid structure is preserved in the glass. 3. Silicate Liquids at High Temperatures In order to extend the understanding to the corresponding liquids, it is essential to investigate the effect of temperature on these speciation reactions, through in situ, high-temperature Raman spectroscopy. This experiment is becoming much more feasible with the availabihty of the rapid and sensitive detection offered by diode-array and CCD devices. In addition, because the desired liquid temperatures are generally above approximately 1000°C, blackbody radiation emitted from the sample and the furnace assembly begins to interfere with the Raman signal in the blue-green region of the spectrum. Because the Raman scattering of silicates is weak, the tail of the blackbody curve can seriously lower the signal-to-noise ratio of the Raman spectrum, rendering it unobservable with conventional Raman techniques. However, micro-Raman spectroscopy, combined with careful spatial filtering before the spectrometer entrance, provides an elegant method for overcoming this problem for in situ, high-temperature investigations of silicate Uquids. Because the incident beam is focused on, and collected from, a |xm-sized region of the sample, blackbody radiation from the remainder of the sample and the furnace assembly is eliminated, especially if a slit or pinhole spatial filter is placed at an intermediate focus before the entrance to the spectrometer. This technique has been used successfully to obtain highquality Raman spectra of molten silicates and aluminates up to approximately 1700°C, as shown in Fig. 26 (Daniel etal, 1992,1993; McMillan etal, 1992b; Mysen and Frantz, 1992b; McMillan, 1993; Neuville and Mysen, 1993; Poe

Figure 25 (a) In situ micro-Raman spectra of an Na2Si409 glass and (b) the proposed compression mechanism (up to 20 GPa), which involves the formation of V- and Vl-coordinated Si from Si-0~ nonbridging oxygens (Q^ species: 1100 cm~^; Wolf et al, 1990b).

350

P. F. McMillan

et al.

A

200

200

NS7

NS5 T,jq: 1255'C Tfl! 945*C

1367*C 820'C 150

150

1440

100

1340

CO

1320

o

C

1268 .1215 165

50

900

1000

1100

800

1200 1300

900

1000

1100 1200

1300

Wavenumber (cm'^J

Wavenumber (cm ^

NS2 T,iq: 870'C Tgi 490'C

NS3 T,jq: 805'C a.

150

800

900

1000

1100 1200

Wavenumber (cm'^)

900

1000

1100 1200

1300

Wavenumber (cm' )

Figure 26 In situ, high-temperature Raman spectra of glasses, supercooled melts and melts along the Na20-Si02 join, as a function of temperature (the values on the right-hand side of each spectrum are in °C). N.B.: Tuq, liquidus temperature; Tg, glass transition temperature, (a) NS7, Tuq = 1367°C, Tg = 820°C; (b) NS5, riiq = 1255°C, rg = 945°C; (c) NS3, Tuq = 805°C, Tg = 445°C; (d) NS2, Tiiq = 870°C, Tg = 490°C. The compositions are abbreviated as NSx:Na20.jcSi02 (Mysen and Frantz, 1992). et al., 1994). Most recently, techniques are being developed to obtain micro-Raman spectra of silicate and related liquids in situ under combined high P-T conditions, using resistance or laser heating in the diamond-anvil cell (Farber and Williams, 1992; Gillet et al., 1993a). This avenue of research

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is a particularly promising one for obtaining information concerning the properties of liquids at depth within the Earth.

4. Interaction of Volatile Species with Melts and Glasses Another area of great interest in the Earth sciences is the interaction of volatile species with aluminosiUcate hquids and glasses. Micro-Raman techniques have played an important role in developing the current understanding of these systems. From near-IR studies on hydrous glasses, Aines et al. (1983) and Stolper and co-workers (1982a,b, 1983) concluded that water is present both as bound hydroxyl groups and as molecular H2O. This interpretation was confirmed by Raman spectroscopy with the observation of a band due to the bending vibration of molecular H2O in hydrous albite glass (McMillan et al, 1983; Mysen and Virgo, 1986a,b). In the system Si02-H20, a band at 970 cm~^ appears with increased water content in the Raman spectrum, which can be assigned to a stretching vibration of SiOH (Q3-OH) units. There is some evidence for a band at lower frequency due to Si(OH)2 units at higher water content, consistent with NMR results (Mysen and Virgo, 1986a; Farnan etal, 1987; McMillan and Holloway, 1987). There is also a peak at 3598 cm~^ in the O—H stretching region which has not yet been assigned to any structural feature (McMillan and Remmele, 1986; Mysen and Virgo, 1986a). Compared with hydrous silica, the O—H stretching bands in hydrous alkah silicate glasses show evidence for much more extensive hydrogen bonding, which may be correlated with the dramatic increase in melt water solubilities along binary alkali-silicate joins (Mysen and Virgo, 1980c, 1986a; McMillan and Remmele, 1986; McMillan and Holloway, 1987). There is controversy over the assignment of the Raman bands in hydrous aluminosihcate glasses (McMillan and Holloway, 1987). In hydrous glasses along the Si02-NaAlSi04 join, a band near 900 cm~^ grows with increasing water content (Myson and Virgo, 1980c, 1986b; McMillan etal., 1983), which McMillan et al. (1983) and Remmele et al. (1986) suggested might be due to Al—OH stretching vibrations. Mysen and Virgo (1980b, 1980c) found no H/D isotopic shift for this band, and concluded that it could not be due to a hydrated species in the glass. However, the lack of an isotope shift for this band could be a result of vibrational coupHng effects (McMillan et al., 1993); thus, it could still be assigned to an Al—OH vibration (Sykes and Kubicki, 1993; Sykes et al, 1993b). There have been several Raman spectroscopic investigations of the dissolution mechanisms of CO2 in silicate and aluminosihcate glasses. Mysen and Virgo (1980a), Rai et al. (1983) and Sharma et al (1979) studied a CaMgSi206 melt composition with up to approximately 5 wt% dissolved CO2, and Verweij et al. (1977) examined potassium siUcate glasses containing

352

P. F. McMillan et al.

carbonate. These investigations showed a strong Raman peak at 1084 cm~^ which grew with increasing CO2 content. This peak is due to the symmetric stretching vibration of CO^T groups in the glass. There was no evidence for bands of molecular CO2, suggesting that all CO2 in the fluid reacted with oxygen in the melt to give C 0 3 ~ and a more polymerized silicate unit. Weak bands near 1430 and 1550 cm"^ were also observed in the spectra. These features are due to the asymmetric stretching vibration (^'3) of COs", which should give rise to a single band of E' symmetry for undistorted carbonate units. The sphtting of the 1^3 band suggests either severe distortion of the col" units in the glass, or the presence of two types of COl~. Mysen and Virgo (1980a,b) and Rai et al. (1983) have also obtained Raman spectra of glasses of NaAlSi308 (Ab) and CaAl2Si208 (An) compositions containing CO2. These authors found evidence for both molecular CO2 and C 0 3 ~ in the Ab glass, but only COl~ units in the An glass. Raman spectroscopy has also been used to examine the dissolution mechanisms of other volatile species, such as H2 and F2, in aluminosilicate Hquids and glasses (Mysen and Virgo, 1985a,b; Luth et al., 1987; Luth, 1988). Pawley et al. (1992) recently used micro-Raman spectroscopy in conjunction with micro-IR techniques to study the dissolution behavior of carbon monoxide in a basaltic melt. The micro-Raman work indicated that no molecular CO was dissolved in the glass, but poorly crystallized graphite was found lining the walls of fluid inclusions in the sample. The fluid inclusions were probed by micro-Raman spectroscopy, and found to contain a CO-CO2 mixture. This method yielded a precise measure of the fluid composition in equilibrium with the glass sample, for which the total CO2 content was determined by micro-IR spectrometry.

ACKNOWLEDGEMENTS We wish to acknowledge support from the National Science Foundation (grants EAR-8916004 and 9219504 and INT-9115888 to PFM; EAR-8920239, 8916754 and 9117858 to RJH) and N.A.S.A. (NAGW-1722 to RJH). PFM also thanks the Institut de Geologic at the Universite de Rennes I (France) for a visiting summer Professorship, during which time part of this review was completed. JD also wants to acknowledge the support from CREGU (GDR CNRS 77) and from a NATO postdoctoral fellowship.

REFERENCES Aines, R. D., Silver, L. A., Rossman, G. R., Stolper, E. M. and Holloway, J. R. (1983). Geol. Soc. Am. Abstr. (with programs) 15, 512.

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8 Biological Applications Michel Truchet

I. INTRODUCTION

Among the numerous approaches to the study of Hving matter, biochemistry, and especially molecular biology, is now prevalent. Many different analytical methods are employed in the field of molecular biology. Although Raman spectroscopy is not the most widely used technique, it is a very important one. Its areas of application are varied; protein conformation, nucleic acid structure, cornea aging, toxic-metal Unking to metalloproteins or nucleic acids and heme structures are some of the subjects treated in an abundant scientific hterature. Biological appUcations of Raman spectroscopy have developed since the invention of the laser and, as in all other fields, they have benefited from continuing technical advances. Examples are the rejection or suppression of fluorescence with the use of counting gates or near-IR excitation and the Fourier transform method. Specific resonance excitation has also been widely used. Recently, the introduction of SERS has yielded spectacular results in biology by considerably enhancing the Raman intensity without the disadvantage introduced by the specific excitation of a chromophore (see Chapter 9). It is not the purpose of this chapter to review this rich subject (Alix et aL, 1985), but rather to point out that this technique in molecular biology is applicable to convenient quantities of substances - samples in the mg to g range. The extraction and purification of suitable samples are, in almost all cases, the first steps in these investigations. The structural basis of Ufe is the cell. Whatever the importance of results obtained on isolated substances, it is essential to place them in the cellular pathways to overview cellular physiology and behavior. Although the largest cells have dimensions of tenths of millimeters (—100 [xm), almost all are in the range 10-20 luim. Therefore, the knowledge at the cellular scale requires the use of a magnifying optical system. Far from being obsolete, microscopy

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is still, and probably will be for a long time, the fundamental technique for studying living matter. It is perhaps appropriate to review the past and present applications of microscopy to biology before considering trends within the perspective of Raman spectroscopy.

II. FROM HISTOLOGY TO CYTOLOGY From the pioneer work of Loewenhook until the 1950s, the discovery of the cellular and tissue structures of animals and plants was dependent on the technical improvements of light-and-glass instruments, commonly called optical microscopes - but perhaps more correctly named photon microscopes. Over a period of a century, the theoretical hmits of photon optics have been nearly reached, namely the diffraction limit of approximately 0.25 |xm, which determines the lateral resolution of images with the use of UV hght and oil immersion objectives. For visible light, the limit is, in practice, approximately 0.5 fxm. Taking into account the keenness of sight, estimated at 0.25 mm for the human eye, the magnifications to be obtained with such instruments is in the range 500-1000. Under these conditions, despite the remarkable inventions of interferential, phase-contrast and polarized light microscopies, it was impossible to observe directly the fine internal structure of cells - or ultrastructures. In spite of these difficulties, the great histologists were able to describe the main components of the cell structure: the nucleus with nucleolus, chromatin and chromosomes, vacuoles and secretion products. Thanks to the photon microscope, the different kinds of cell and their relationship in tissues, as well as cell division with the formation and separation of the chromosomes, were known and the concept of cell differentiation was acquired before the end of the last century. On the other hand, the existence of the Golgi apparatus, the mitochondria, the Balbiani rings or the centrioles was a question which debated until the 1950s. The development of the electron microscope, aided by a considerable amount of work on the techniques of sample preparation for this new instrument, made possible the description of the morphology of the cell at the ultrastructural level. With a resolution enhancement of nearly a thousand and subsequent magnification of several hundred thousands, molecular dimensions were reached. Not only was the reahty of the Golgi apparatus, of mitochondrias, and of centrioles definitely established, but unsuspected internal structures were also discovered. The three layers of the limiting membrane, the two membranes of the mitochondrial wall, the complex system of internal membranes or endoplasmic reticulum and, finally, the lysosomial structure were observed, discussed, confirmed. At last, their functional significance with regard to cell physiology was understood (Darnell et al, 1988).

Biological Applications 369 III. FROM MORPHOLOGY TO ANALYSIS The determination of the physiological role of each cell part has been made in almost all cases by comparison of the results of biochemical investigations with those of morphological observations. Various techniques, e.g. centrifugation, have been used to extract a given cell component, such as mitochondrias. However, the microscope is not capable of yielding knowledge of the chemical composition of a cell structure or determining the chemical nature of the product of the activity of a given structure, such as the Golgi apparatus. Yet, as far back as the middle of the last century, histologists such as Raspail undertook the chemical analysis of the cell (Pearse, 1972). Thanks to special reactions adapted to the (xm size of the substances to be identified, histochemistry was born (Gabe, 1964; Martoja and Martoja, 1967; Ganter and Jolles, 1970). It was almost simultaneously in the United States and in France that Tousimis (1963) and Galle (1965), respectively, obtained the first true histophysical results. Unfortunately, perhaps, one century later, histochemistry is now not as efficient as biochemistry. For elemental analysis there are numerous identification reactions, but few are both sensitive and specific. However, Perl's method is excellent for iron, while Morin's fluorescent probe is useful for aluminium at sufficiently high concentrations. In almost all methods there is a lack of sensitivity or an insufficient specificity, or both. In other cases, such as the excellent von Kossa reaction for calcium, it is the hgand, or the anion, which is actually detected. For molecular analysis, reactions are available to characterize a given functional group, e.g. S-S, SH, or 1-2 glycols. Specificity tests, such as reversible methylation or enzymatic digestion, do not exist for all cases. Immunological reactions are more specific, but require the induction of the immunological reaction in question. Finally, tissues and cells have to be fixed chemically, or by freezing, before the observation, which excludes the study of the reaction dynamics. Then, despite the great number and variety of reactions, and the work of the masters of histo- and cytochemistry such as Pearse, Gabe and Thierry, analysis under the microscope is not as efficient as morphological investigation. Fully aware of this problem, some histochemists such as Caspersson, Policart and Lison tried to develop analytical methods involving a general physical principle, such as emission spectrometry (Lison, 1960). But, in the 1930s, the technology was not sufficiently developed to yield successful results from these efforts. It was almost simultaneously in the USA and in France that Tousimis and Galle obtained in 1963 and 1965, respectively, the first true histophysical results (Galle, 1965; Tousimis, 1963). These data were obtained with the Castaing electron microprobe (Castaing, 1951), new at that time, which was

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applied to the elemental analysis of volumes at the micrometric scale in histological sections observed through the photon microscope. Ballan-Dufrangais and Martoja (1971) compared the performances of the electron microprobe with the results of histochemical analyses. They concluded that this instrument was better for the elemental analysis of almost all chemical reactions. During the course of the following decade, rapid technical improvements led to a new generation of instruments in which the electron microprobe was coupled to an electron microscope, allowing the analysis of sub-|xm structures, localized at the ultrastructural level, mainly with the use of the transmission electron microscope (TEM). Analyses were also performed on samples observed by surface-scanning electron imaging (SEM). This 20-year period was also that of the invention and appHcation to biology of numerous other analytical microscopic methods, e.g. secondaryion mass spectrometry (SIMS) (Castaing and Slodzian, 1962; Truchet, 1982), laser mass spectrometry (LMS) (Hillenkamp et aL, 1975; Eloy, 1980), electron energy-loss spectrometry (EELS) (Castaing et al., 1965; Colliex, 1984) and laser Raman vibrational spectroscopy (Delhaye and DhameUncourt, 1975). Certain other techniques, such as Auger spectroscopy, have still not been appUed to the investigation of biological systems. Among these various methods, only EELS can carry out an analysis at the ultrastructural level, while Raman spectroscopy is the only truly molecular analytical method. The invention of the Raman microprobe by Delhaye and Dhamelincourt (1975) has allowed vibrational molecular analysis to be performed on volumes of only a few ^xm^ (~10~^^ g), localized through the photon microscope (see Section IV). There is promise that the ultrastructural level will soon be reached (see Section V). IV. RAMAN MICROANALYSIS APPLIED TO BIOACCUMULATIONS The final product of cellular metabolic activity is often concentrated into special structures, generically named bioaccumulations. They are stored inside the cell or rejected outside, in the extracellular compartment. Their structures and compositions are diverse. In plant cells they are often vacuolar, whereas three main forms can be distinguished in animal cells. A. Lysosomes In both vertebrate and invertebrate cells, De Duve discovered in the 1960s the existence of small structures rich in acidic enzymes. These are called lysosomes, because their main function is catabolic. At cell death, autolysis is carried out by the digestive enzymes contained in these structures. During

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life, their essential function is to scavenge the cytoplasm for rejected or dangerous substances. If their complete degradation is not possible, these substances are stored into the envelope of the lysosomes and in this way are isolated from the metaboUc pathways. Specific reactions can also occur in the lysosomes, such as the precipitation of toxic elements as nonbiodegradable complexes. In many invertebrate species lysosomes play an important role in the digestion of nutrients. In species in which the evolution of the physiological excretory function is not achieved, the continuous catabolism of worn molecules, without excretion, generates significant quantities of a particular substance. This substance accumulates with the aging process. At the initial stage of an ecological study, biological indicators of pollution were identified by analytical methods such as atomic absorption spectrophotometry performed on dissected organs, or on the entire body. Under these conditions it is not possible to distinguish a natural tissue overload from the consequence of a pollution. Only analyses performed at the cellular scale, e.g. analytical microscopic methods such as EPMA, SIMS and Raman spectroscopy, lead to the identification of the bioaccumulation mechanism, and thus clearly distinguish between natural processes and detoxification reactions. For example, Martoja and Berry (1980) demonstrated that considerable amounts of the very toxic metal, Hg, can be accumulated by marine organisms. However, this process has no dangerous consequences, as the metal is precipitated as the nonbiodegradable complex HgSe. Analogous results have been demonstrated for silver, for the precipitate Ag2S (Martoja et al., 1988). These results, which are of interest in the analysis of food chain contamination, are among the most important contributions of analytical microspectroscopy to the investigation of biological systems. Raman microspectrometry is of particular importance in the appUcations described above, as it is the only method which can estabhsh the molecular identity of the sample. Furthermore, the risks involved in chemical extraction and purification are thus avoided. In the present example, because lysosomes are scarce, biochemical methods are impractical. Another example is that of the edible mollusc Littorina, in which oxygen is transported into the blood by the copper protein, haemocyanin. When rejected after use, this molecule is not completely degraded and is excreted in the urine, as is vertebrate haemoglobin. Copper is stored in the lysosomes of a particular type of cell, the pore cells. The subsequent increasing copper content of these animals with age was considered to be an indication of the pollution of sea-water. Thanks to the Raman microscope, the symmetric vibrational mode of CuS was recorded at 474 cm~^ by direct observation of a pore cell, despite a strong fluorescence (Martoja et al., 1980). This result demonstrated that copper accumulation is a natural physiological process, rather than the result of pollution.

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B. Spherocrystals Spheroidal structures, ranging from 0.1 to 10 ixm in size, are frequently observed in some tissues of invertebrate animals. These structures are composed of concentric layers around a central nucleation point which originates from an ergastoplasmic cisternae. These structures are usually called 'spherocrystals' (Ballan-Dufrangais, 1975). Spherocrystals occur naturally and harmlessly in invertebrate tissues, but in vertebrates they occur as a result of a pathological process and are called kidney stones. In invertebrates they are involved in normal physiological regulations, such as the temporary storage of reserves, e.g. calcium salts for the mineral precipitation of skeletal structures. They can act as a kidney accumulation, like lysosomes, but their detoxication role is very important. These structures are found to coexist in numerous cell types (Jeantet, 1981). When they are abundant, they can be extracted and studied biochemically; their function can then be established. But when they are scarce, especially in a diffuse interstitial tissue, only microanalytical methods are available. Although EPMA and SIMS can give the elemental composition of these structures, only Raman microscopy can determine their molecular composition (Ballan-Dufrangais et aL, 1979). In gastropods (molluscs) there has been debate as to the exact composition of the numerous spherocrystals, which are sparse in conjunctive tissues. However, Raman microscopy has shown that they are composed of calcium salts - not only carbonates, as is normal for invertebrates, but also phosphates (Martoja and Truchet, 1983). Generally speaking, the spherocrystals are ideally suited to Raman microscopic studies. As their size is on the (xm scale, the significant concentration of these substances in the layers and their resistance under laser illumination are favourable parameters. The detection of calcium and phosphorus by EPMA or SIMS (or EELS) in spherocrystals is only an indication of the presence of phosphates. In fact, calcium-binding proteins which are rich in phosphorus are known. Only Raman microscopy, by the observation of the symmetric vibrational mode at 965 cm~^, can reveal the mineral composition. In the case, which is most common in invertebrates, where the calcium salt is a carbonate, the crystalline structure can be determined, and the presence of vaterite, calcite or aragonite can be established (Truchet et al., 1989). The last point is of particular importance because, by determining crystalline structures in samples from ancient or modern times, Raman microscopy can contribute to the study of the evolutionary process. Spherocrystals do not only contain minerals - they also contain entirely organic concretions produced by the catabolism of nucleic acids, which also yield excellent Raman spectra. In Blatella, a well-known insect (the cockroach), biochemical studies of the very abundant spherocrystals of body fat and the genital gland (utricles) determined a mixture of uric acid and Na

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and K urates (Ballan-Dufrangais, 1975). Uric acid is prevalent in body fat, whereas urates are more abundant in the utricle. The interesting question was then whether these different substances developed in different spherocrystals. A Raman microscopic study confirmed the difference in composition between the tissues, but demonstrated clearly that the population of spherocrystals was homogeneous (Ballan-Dufrangais et al., 1979). C. Secretion Products (Chitin)

It is well known that, in the phylum Arthropodia, the external skeleton is made of an organic polymer known as chitin. The monomer of chitin is A^-acetyl-glucosamin, but associated proteins play an important structural role and are very difficult to separate in the chitin purification process. Some speciaHsts consider that truly purified chitin does not exist (Gooday, personal communication). In the past the pesticide DDT was widely used against insects, but its toxicity was such that it is now banned. To replace it, less dangerous substances have been tested, among which diflubenzuron, commercialized by Philips-Duphar over the past 15 years, seems promising. It acts on moulting by disturbing the synthesis of the new cuticle. However, serious discussions still occur about the true mechanism of its action, as well as its secondary effects on insects and the ecosystem (Truchet et at., 1981). To understand better the action mechanism an effort was made to characterize chitin and its precursors directly inside the insect epithelial cell with the use of the Raman microscope (Truchet and Mauchamp, 1986). Although it is possible (but very time-consuming) to obtain chitin spectra from the cuticle in histological sections, the assignment of peaks to structural characteristics is much more difficult and questionable than might be presumed, especially with respect to the precursors whose Raman spectra are very similar to that of chitin. However, in the initial experiments it was found that the signal-to-noise ratio obtained in these spectra with the use of the Raman microscope was insufficient. This problem is being re-examined with the new generation of micro-Raman instruments now available in the hope of obtaining spectra of chitin precursors in the insect cell.

V. TRENDS IN RAMAN MICROSCOPY AS APPLIED TO CELLS AND TISSUES A. Living Samples

Among the principal advantages of the photon microscope is its ability to operate in air. Thus it has been used to observe living organisms from the

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date of its invention. The living sample may be an organism, animal or plant, occasionally doped or mechanically confined; recent developments in cell culture have opened a new field in the microscopy of living samples. The medium is normally not perturbed by the observation, and thus the physiological state of the cell is preserved. However microorganisms, because their size is at or below the limit of the resolution of the photon microscope, are not suitable living samples for analysis by photon microscopy. The direct analysis of living samples with the use of Raman microscopy was exploited early in its development (Arrio etal., 1980) and, more recently, spectacular results have been obtained on cultured cells by application of SERS (see Chapter 9). The possibility of obtaining SERS spectra routinely from living matter or histological sections is probably the most exciting trend in the biological application of Raman microscopy. Another great advantage of Raman microscopy is the spatial improvement offered by the confocal configuration, as recently exploited by Puppels et al. (1990). These authors also showed that red laser fight was not as destructive as green or blue light. They were thus able to obtain spectra from living ceUs under good physiological conditions (Puppels et al., 1991). Their spectra of nucleic acids in situ are of particular interest. They point out the great similarity of all of the spectra, independent of the laser-probed area in the nucleus. This observation is surprising, because the morphology of the nucleus is fundamentaUy heterogeneous, as observed with both photon microscopy and electron microscopy. Some biologists, including Dubochet et al. (1992), hypothesize that the living nucleus is homogenous and that its classical morphology is an artifact. This debate on the internal structure of the living nucleus is fundamental in biology. In biochemistry, as well as in the domains of genetics and evolution, it would be a great contribution of Raman microscopy if the above results were confirmed and generalized.

B. Ultrastructures One of the principal disadvantages of the photon microscope is its resolution fimitation of approximately 0.5 |xm, particularly when the resolution required to observe the internal structure of the cytoplasm organelles is ten to several hundred times smaller than this value. Since the 1960s the contribution of electron microscopy to the knowledge of the cell function has been considerable. It is unfortunate that Raman microscopy is fimited to the structural level in cell physiology and thus cannot provide ultrastructural information. Other analytical microscopic methods such as EELS or EPMA are effective routinely at high magnifications, e.g. 5000, 10 000 or more. Although the results of EELS analysis in biology are scarce, published results obtained with EPMA are numerous and varied. In the field of environmental pollution, Ballan-Dufrangais et al. (1980) used

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EPMA to identify toxic metals with dimensions as small as 0.05 |xm accumulated in lysosomes. With this method only elements are detected, but semiquantitative analysis yields values for the atomic ratios, such as P/Ca = 1, Ag/S = 0.5 or Cu/S = 0.33 and thus hypotheses can be established concerning the molecular nature of the bioaccumulation, e.g. calcium phosphate, silver sulfide (Ag2S) or metallothionein (Jeantet et al., 1980). These hypotheses cannot yet be verified, as direct molecular analysis is lacking at the ultrastructural level, although the above-mentioned molecules have welldefined Raman spectra. To obtain these spectra it would be necessary to couple Raman microscopy with electron microscopy and microanalysis (Truchet and Delhaye, 1988). The conditions of this coupUng and the state of the art in this area are detailed in Chapter 5 of this volume. The determination of the nature of molecular ligands of toxic metals is not an academic question. The pollution of the biosphere is becoming serious and our knowledge of cellular detoxication processes is not advancing as rapidly as the contamination. As previously emphasized, analytical microscopy provides a promising set of tools with which to increase our knowledge of the defence mechanism of organisms. The ability to reach the ultrastructural level in the biological applications of Raman microscopy is thus not so much a technological challenge as an ecological necessity.

VI. CONCLUSION

This brief survey of the present and potential appHcations of Raman microscopy to the investigation of cells and tissues is not a review, but rather is an attempt to place this analytical method in the general perspective of the contributions of microscopy to biology. At the present time, the impact of Raman spectroscopic analysis in biology is not as important when it is performed through a microscope as when it is appHed in biochemistry to the investigation of extracted and purified substances. This point was estabHshed at the beginning of this chapter. However, the considerations which were subsequently presented concerning its perspectives demonstrate clearly that Raman microscopy is important in this field, although it is at present richer in promises than in results. There is a reasonable hope that Raman microscopy will reach the level of performance of macro-Raman spectroscopy in the early years of the twenty-first century, and perhaps even surpass that mark. In 1985, SERS was unknown; yet just 10 years later it has become effective at the microscopic level, as shown in the following chapter. The importance of red-light excitation - as well as near-infrared excitation - in reducing fluorescence and in protecting living samples, has been demonstrated. The possibility of obtaining spatial resolution at the ultrastructural level

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is promising. To reach the efficiency of macroscopic R a m a n spectroscopy is the present challenge to R a m a n microscopy. It can thus be safely concluded that the latter technique has now become a specialty in its own right.

REFERENCES Alix, A. J. P., Bernard, L. and Manfait, M. (eds) (1985). Spectroscopy of Biological Molecules. John Wiley & Sons, Chichester. Arrio, B., Dupaix, A., Fresneau, C , Lecuyer, B. E. and Volfin, P. (1980). Uactualite chimique 4, 19. Ballan-Dufranqais, C. (1975). Doctoral thesis, Universite Pierre et Marie Curie, Paris. Ballan-Dufrangais, C. and Martoja, R. (1971). J. Microsc. 11, 219. Ballan-Dufrangais, C , Truchet, M. and Dhamelincourt, P. (1979). Biol Cell. 36, 51. Ballan-Dufrangais, C , Ruste, J. and Jeantet, A. Y. (1980). Biol. Cell. 39, 317. Castaing, R. (1951). Doctoral thesis, ONERA, Paris. Castaing, R. and Slodzian, G. (1962). / . Microsc. 1, 395^10. Castaing, R., ElhiH, A. and Henry, L. (1965). C.R. Acad. Sc. Paris 261B, 3999. CoUiex, C. (1984). Proc. 8th Eur. Con. Electron Micro., Budapest. Darnell, J., Lodish, H. and Baltimore, D. (1988). Biologic moleculaire de la cellule, 2nd edn. De Boeck-Wesmael, Brussels. Delhaye, M. and Dhamehncourt, P. (1975). J. Raman Spectrosc. 3, 33. Dubochet, J., Bednar, J., Furrer, P. and Stasiak (1992). Proc. 32th Coll. SFME, SFME Pub., Rouen, France, p. 3. Eloy, J. F. (1980). Proc. 5th Int. Symp. High Purity Mat. Sci. Technol. DDR Akad. Wiss., Dresden. Gabe, M. (1964). Techniques histologiques. Gauthier-Villars, Paris. Galle, P. (1965). Doctoral thesis, L'Expansion, Paris. Ganter, P. and Jolles, B. (1970). Histochimie normale et pathologique. GauthierVillars, Paris. Hillenkamp, F., Kaufmann, R., Nitsche, R. and Unsold, E. (1975). Appl. Phys. 8, 341. Jeantet, A. Y. (1981). Doctoral thesis, Universite Pierre et Marie Curie, Paris. Jeantet, A. Y., Ballan-Dufrangais, C. and Ruste, J. (1980). Biol. Cell. 39, 325. Lison, L. (1960). Histochimie et cytochimie animales. Gauthier-Villars, Paris. Martoja, R. and Berry, J. P. (1980) Vie et Milieu 30, 7. Martoja, R. and Martoja, M. (1967). Initiation aux techniques de l'histologic animale. Masson, Paris. Martoja, M. and Truchet, M. (1983). Malacologia 23, 333. Martoja, M., Vu Than Tue and Elkaim, B. (1980). /. Exp. Mar. Biol. Ecol, 43, 251. Martoja, R., Ballan-Dufrangais, C , Jeantet, A. Y., Gouzerh, P., Amiard, J. C , Amiard-Triquet, C , Berthet, B. and Baud, J. P. (1988). Can. J. Fisheries Aquat. Sci. 45, 1827. Pearse, A. G. E. (1972). Histochemistry, Theoretical and Applied. ChurchillLivingstone, London. Puppels, G. J., De Mul, F. F. M., Otto, C , Greve, J., Robert-Nicoud, M., Arndt-Jovin, D. J. and Jovin, T. M. (1990). Nature 347, 301.

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Puppels, G. J., Garritsen, H. S. P., Segers-Nolten, G. M. J., Demiil, R F. M. and Greve, J. (1991). Biophys. J. 60, 1046. Tousimis, A. J. (1963). Proc. Vllth Int. Conf. Electron Probe Micro-Anal. John Wiley, San Francisco, p. 45. Truchet, M. (1982). Doctoral thesis, Universite Pierre et Marie Curie, Paris. Truchet, M. and Delhaye, M. (1988). / . Microsc. Spectrosc. Electron. 13, 167. Truchet, M. and Mauchamp, B. (1986). In: MuzzareUi, R. A. A., Jeuniaux, C. and Gooday, G. W. (eds), Chitin in Nature and Technology. Plenum Press, London, p. 3. Truchet, M., Grasset, M. and Vovelle, J. (1989). Actes Symp. GEORAMAN-89. Univ. Toulouse Pub., p. 27. Truchet, M., Lauverjat, S., Lamy, M. and Denneulin, J. C. (1981). Pesticide Biochem. Physiol. 15, 253.

Applications in Medicine Michel Manfait and Igor Nabiev

I. INTRODUCTION Raman scattering is a vibrational spectroscopic technique that can both fingerprint components of biological and biomedical species and identify polymorphs of crude mixtures, extracts, cells and chromosomes (Spiro, 1987). By coupling an optical microscope to a conventional Raman spectrometer, the technique becomes a microprobe with a spatial resolution of less than 1 juim, determined by the wavelength of the radiation and the numerical aperture of the microscope objective (cf. Chapter 2). The first part of this review will be devoted to conventional Raman microscopy. It will summarize Raman microscopic studies of some biological tissues, ocular lenses, living cells and chromosomes. The fundamental contributions in this field have been made by the groups of J. Greve (University of Twente, The Netherlands) and N.-T. Yu (Georgia Institute of Science and Technology and The Hong Kong University). The second and major part of the review will summarize a relatively new field of Raman microspectroscopy - the application of the surface-enhanced Raman scattering (SERS) effect to the microscopic investigation of biomedical species. Approaches to the preparation of SERS-active surfaces optimized for biomedical samples and the possibilities of increasing SERS selectivity will be discussed. SERS microspectroscopic studies of intact biological organisms, model systems and living cells, including living cancer cells treated by drugs, will be presented as examples.

A. Conventional Raman Microscopy The first Raman microprobe was developed in the 1970s (Delhaye and Dhamelincourt, 1975; Rosasco et al., 1975). The difficulties which arise in

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the application of Raman microprobe spectroscopy in biomedicine are basically the same as those encountered in conventional Raman spectroscopy. They include the small Raman cross-section, which results in very low signal levels, the possibility of sample damage, and the strong fluorescence background which is often observed in the spectra of crude biomedical extracts and mixtures, tissues and cells. The problem of sample damage can often be solved by optimizing the experimental conditions (see Section II), the spinning of solid samples or the flowing of liquids, cooling (to avoid thermal damage), and the use of inert environments, e.g. vacuum or nitrogen (to avoid photo-oxidation). The inherent low efficiency of Raman spectroscopy can be compensated in two ways. One way is to improve the sensitivity of the instrumentation. This development was recently made in the laboratory of Professor J. Greve (Puppels et al., 1990a,b; 1991). The main features of a very refined, confocal Raman microspectrometer (Fig. 1), which are briefly summarized below (Puppels et aL, 1990a; 1991), are: (i) High signal throughput from microscope objective to detector (40%), (ii) Sensitive, essentially photon-noise-limited, signal detection by means of a liquid-nitrogen-cooled CCD camera (quantum efficiency 40% at 700 nm, 10 electrons of read-out noise per channel, negUgible dark current), (iii) Confocal signal detection, leading to an effective suppression of the background Raman signal from substrates or buffers (with a 1.2 NA objective and a 100 jxm pinhole, spatial resolution of 0.45 X 0.45 X 1.3 |xm^) and (iv) The use of laser light of wavelength 660 nm to prevent radiation damage of samples. Another way to improve instrument performance is to enhance the Raman signal. To study nucleic acids and proteins, it is necessary to use UV excitation. With the use of a recently developed UV Raman microspectrometer, it was shown that single-cell, resonance Raman spectra can be obtained (Sureau et al., 1990). However, the increased danger of UV radiation-induced damage should be noted. Yet another approach is SERS microspectroscopy, which is currently being employed in this laboratory* (Nabiev et «/., 1988, 1991b, 1993, 1994a,b; Nabiev and Oleynikov, 1990; Manfait et al., 1992a,b; Morjani et al, 1993; Sokolov etaL, 1994a,b; Sharonov etal, 1994a,b; Feofanov etal, 1995). This development represents a new technique in the study of biomedical samples.

*Laboratoire de Spectroscopie Biomoleculaire, UFR de Pharmacie, Universite de Reims, 51, rue Cognacq-Jay, 51096 Reims, France.

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Grating

M (concave) Charge-coupled device camera

Figure 1 Confocal Raman microspectrometer (CRM). The laser light of 660 nm wavelength from a DCM-operated model 375B Spectra Physics dye laser is focused on the object under investigation by a microscope objective with high numerical aperture. The light travels through a pinhole, which enables confocal detection (Brakenhoff et al., 1979). Lateral spatial resolution in the CRM is determined by the laser focus dimensions and is <0.5 luim. A pinhole of 100 jxm diameter gives a depth resolution of 1.3 ixm. The spectrometer consists of a chevron-type dielectric, bandpass filter set, which combines efficient stray light suppression (by a factor of 10^) with a high transmission (80-90% in the spectral interval 600-1800 cm"~^) for Raman light (Puppels etaL, 1990a) and a wavelength dispersion stage. A liquid-nitrogen-cooled, charge-coupled device camera (equipped with an Enghsh Electric Valve Co. P8603 B charge-coupled device chip, Wright Instruments, Ltd) is used for signal detection. It combines a high quantum efficiency (c. 40% at 700 nm) with almost noise-free operation (negUgible dark current and an r.m.s. read-out noise of 10 electrons, equaUing 10 detected photons). The spectral resolution of the microspectrometer is approximately 6-7 cm~^. Abbreviations: M, mirror; L, lens.

B. Surface-enhanced Raman Scattering and the SERS Microprobe Surface-enhanced Raman scattering spectroscopy, which has developed relatively recently, offers the possibility of circumventing many of the problems of conventional Raman spectroscopy, as well as in its microscopic applications. Molecules adsorbed on metal surfaces are found to have an unusually large Raman cross-section under certain conditions (for reviews of this field, see Nabiev et al., 1988, 1993, 1994b; Nabiev and Manfait, 1993). In this phenomenon the optical properties of molecules in the vicinity of the

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solid surface are modified. The magnitude of the Raman scattering crosssection is usually enhanced, to an extent which depends on the chemical nature of the adsorbed molecules, the type of metal surface and its structure. The greatest enhancement occurs for molecules adsorbed on silver, gold, or copper. The metal surface must be rough and have specific adsorption properties. This technique has been found to be very useful in increasing the spectral sensitivity, and thus yielding new information on both organic and inorganic molecules (Chang and Furtak, 1982). The SERS effect is accompanied by strong quenching of fluorescence, providing the possibility of extending the range of molecules and crude mixtures and extracts that can be investigated. The ultrahigh sensitivity of SERS facilitates the measurement of spectra at concentrations down to 10~^^moll~\ which allows the laser power to be significantly reduced. Simplified and much less expensive minispectrometers can then be employed (Nabiev et al., 1988, 1993). There are at least two major types of mechanism that contribute to the SERS phenomenon: an electromagnetic effect associated with large local fields due to resonances occurring in the microstructures on the metal surface, and a chemical effect involving a scattering process associated with chemical interaction between the molecule and the metal surface (Otto et al., 1992). Electromagnetic enhancement mechanisms are characterized by the following features: (i) The effects are long range (compared to the length of a chemical bond), since the dipole fields induced in polarizable metal particles vary as the inverse cube of the distance to the center of a given particle. (ii) The electromagnetic effects are generally independent of the nature of the adsorbed molecule. (iii) The enhancement depends on the electronic structure of the substrate and the roughness of the surface, since the frequencies of the surfaceplasmon resonances depend on these factors (Chang and Furtak, 1982). The chemical effect is associated with the overlap of metal and adsorbate electronic wavefunctions, which leads to ground-state and light-induced charge-transfer processes. The contribution of the chemical effect to SERS is necessarily short range (1-5 A). This mechanism depends on the adsorption site, the geometry of binding, and the energy levels of the adsorbate molecule. Chemical enhancement can provide useful information on chemisorption interactions between a metal and an adsorbate. However, this enhancement is not a general mechanism and is restricted by its chemical specificity (Otto et al., 1992). The instrumentation required for SERS is relatively simple and similar to that used in conventional Raman spectroscopy. Maximal Raman enhancement can be achieved, as a rule, with the use of Ag, Cu or Au surfaces. Thus

Applications

in Medicine

383

far, SERS experiments have been carried out mainly on Ag or Au. Excitation wavelengths in the red or near-IR regions are required to observe SERS on Au and Cu surfaces, whereas either visible or near-IR excitation may be used on Ag (Boyd et aL, 1984). Electromagnetic field calculations show the existence of an excitation wavelength threshold for Au and Cu at c. 600 nm (Van Duyne, 1979). The main advantage of the application of SERS to problems in chemistry, biochemistry and biomedicine is the possibiUty of making measurements which preserve the normal conformation of the molecule or supramolecular complex. Some research teams (Smulevich and Feis, 1986; Nabiev et al., 1993) have published data which demonstrate that an interaction with the silver surface in silver hydrosols is very weak and, thus, does not disturb the structure of the molecules or their interactions within the complexes. On the other hand, significant perturbations to the structure of hem-containing proteins have been found in some SERS-active systems (Smulevich and Spiro, 1985). The difficulty in obtaining an independent control of the influence of a silver (or in general metallic) surface on a biomolecular structure is due to the lack of 'surface-enhanced' methods other than SERS spectroscopy. Hence, it is quite easy to lose the signal from the molecular layers located in the vicinity of the surface when the strong background signal from the molecules in the bulk is detected by traditional (normally non-'surfaceenhanced') methods. It would seem to be advantageous to use SERS spectra to control the molecular structure in the vicinity of the metal surface by comparison with the corresponding Raman (or resonance Raman) spectrum. In practice this procedure is not simple, as SERS spectra usually differ markedly from the corresponding Raman spectra of free molecules due to changes in selection rules and the short-range character of the Raman cross-section enhancement in the SERS-active systems. These effects are manifested in the selective enhancement of certain vibrations, as well as in the appearance of new bands in a SERS spectrum (Otto et al., 1992). Moreover, the Raman spectra are obtained at concentrations of at least three orders of magnitude higher than those used in the observation of the SERS spectrum of the same compound. Furthermore, the former usually have a significant contribution from parasitic fluorescence. Finally, very few examples of close correlation between SERS and Raman spectra of the same biological compound are known from the literature (Morjani et al., 1993; Nabiev et aL, 1993). Recently, Nabiev et al. (1994d) reported a detailed comparative resonance Raman and SERS study of mitoxantrone, a highly potent anticancer drug currently used in chnical trials of non-Hodgkin's lymphomas, acute myeloid leukaemias and advanced breast cancer. The evaluation of the influence of the surface of the silver hydrosol on the molecular interactions within the drug/DNA complexes was made by means of comparison of the resonance

384

M. Manfait and I. Nabiev

Raman and SERS spectra of free mitoxantrone (in water solution and in deuterium oxide) and of its complex with DNA. Interactions between the drug and DNA induced identical changes in the SERS and resonance Raman spectra of the drugs. The data showed that an adsorption of the drug/DNA complex on the surface of the silver hydrosol does not induce detectable perturbations of the molecular interactions within the complexes. The idea of coupling the Raman microprobe with SERS was originally proposed by Van Duyne et al. (1986). Calculated detection limits were less than 1 amol in the probe beam, which corresponds to 10^ molecules, and were as few as c. 600 molecules (Taylor et al., 1990) for compounds excited in the region of an electronic transition (surface-enhanced, resonance Raman effect). These detection limits show that the coupling of SERS and Raman microprobe spectroscopy is a very promising technique for molecular trace analysis. A wide range of experiments designed to probe structure, topology and composition of biomedical species with the use of SERS microscopy can readily be envisioned if, indeed, the measurements are feasible. Some of the experiments which are currently being carried out in this laboratory are: (i) Determination of the distribution of drugs inside and on the membrane of a living cell (Nabiev et al, 1991a,b, 1993; Manfait et al., 1992a,b; Sokolov et al. 1992; 1993b; Morjani et al., 1993). (ii) Studies of cell membrane components (Nabiev and Manfait, 1992; Sokolov et al, 1992, 1993b). This SERS experiment includes the possibility of observing the membrane constituents which can interact with the surface of SERS-active substrates (siahc acid residues, terminating polysaccharide chains of membrane glycoproteins, membrane-bound targets for antitumour drugs, etc.). (iii) Development of new types of SERS-active substrates which are applicable to the analysis of biomedical samples (Nabiev et al., 1988, 1993; Nabiev and Oleynikov, 1990; Oleynikov et al, 1990; Sokolov et al, 1992).

II. EXPERIMENTAL CONDITION FOR THE RAMAN MICROPROBE ANALYSIS OF BIOMEDICAL SAMPLES As noted above, the problem of sample damage is very important in Raman and SERS-microprobe applications. It is even more serious for very delicate biomedical preparations. A number of experiments have been carried out in an effort to elucidate the mechanism of sample degradation (e.g. Calmettes and Berns, 1983; Ashkin and Dziedzic, 1987; Coohill et al, 1987).

Applications in IVIedicine 385 A. Conventional Raman Microprobe Technique

Puppels et al. (1991) narrowed the range of potential mechanisms of damage to biological samples and optimized the experimental conditions for studies of single living cells - chromosomes, granulocytes, etc. They used high numerical-aperture (N.A.) objectives which focus the laser light into a spot of c. 0.5 |xm in diameter. This application leads to high Ught intensities, e.g. an irradiance of 0.25-10 MWcm~^ for 0.5-20 mW of laser power. A degradation of cells and chromosomes under the influence of 514.5 nm light was observed at all laser powers tested. A bleaching of the sample (Fig. 2) was accompanied by a gradual decrease in the intensity of the Raman signal. It is interesting to note that for a model solution of isolated DNA and histone protein irradiated for 3 h with a laser power of 25 mW (12.5 MW cm~^) at 514.5 nm, no signs of sample degradation were found. No bleaching effects for cells and chromosomes were observed with the use of laser light of 632.8 or 660 nm. The percentage of cells (freshly isolated human lymphocytes) surviving a 5 min irradiation with laser light was determined as a function of laser power and wavelength (Puppels et al., 1991). The results are shown in Fig. 3. It is clear that laser Hght of 632.8 or 660 nm wavelength is much less harmful than that at 514.5 nm. On the other hand 514.5 nm irradiation already affects cell viability at a laser power of 5 mW. However, no adverse effects are noticed with 632.8 or 660 nm irradiation for laser powers up to 20 mW. Hence, sample damage can be avoided with the use of laser light of longer wavelengths. Puppels et al. (1991) carefully analyzed the problem of sample degradation and excluded the possible contribution of multiphoton absorption processes, as well as sample and substrate heating. They concluded that the amount of damage depends only on the total laser hght dosage and not on its intensity. One-photon laser light absorption by chromosomes and cells is thus the only possible process involved in sample degradation. The fact that CT-DNA and histone, two of the main chromosome constituents in their purified form, are not susceptible to radiation damage (even at 514.5 nm), shows that other compounds acting as photosensitizers must be present in chromosomes and cells. The sensitizer (e.g. flavins in the cell enzymes) does not absorb Hght in the red region because an excitation of the spectra with laser hght of 660 nm does not induce sample degradation. Green laser excitation takes the sensitizer via the singlet state into a long-lived triplet state. It can then react with an oxygen molecule to form a singlet oxygen or a superoxide ion. The reactive oxygen species could cause the observed radiation damage through oxidation of the DNA bases or amino acids, leading to lesions in these molecules. In conclusion, to avoid degradation of biomedical samples the use of red excitation for recording conventional Raman microprobe spectra or the deposition of the samples in inert media (if shorter-wavelength excitation is necessary) is strongly recommended.

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Figure 3 Percentage of cells (lymphocytes) surviving a 5 min laser light irradiation as a function of laser light power at wavelengths of 514.5 nm, 632.8 nm and 660 nm: O 514.5 nm, objective: 63x Zeiss Plan Neofluar water immersion (N.A. = 1.2) focus diameter: c. 0.5 |xm; • 632.8 nm, objective: 40X Nikon E Plan (N.A. =0.65) focus diameter: c. 1 |jim; D 660 nm, objective and beam diameter. The laser beam was focused in the center of the cells. Each point on the graph represents 10 irradiated cells (20 for the 660 nm 25 mW point). Control samples contained > 90% living cells.

B. SERS Microprobe Technique

No significant sample degradation was observed in the SERS experiments with water-soluble and membrane proteins, nucleic acids or cells (Nabiev et a/., 1988, 1993; Nabiev and Manfait, 1992). Once a stable SERS-active medium has been formed, direct photo thermal or photochemical damage is minimal, even under excitation in the green, blue or violet regions. This result can be explained by the very great Raman cross-section enhancement, especially for compounds with electronic transitions in the visible region. Hence, laser powers of the order of 1 (xW (irradiance of 0.5 kW cm~^) have been used routinely in the SERS microprobe experiments carried out in this laboratory (compared with c. 5 MW cm~^ employed in conventional Raman microscopy). Thus, the light intensity used in the SERS microprobe is approximately 10000 times less than that of the conventional Raman microprobe. SERS-active substrates used with the Raman microprobe, such as hydrosols, island films, nuclear pores, etc. (see Section V. A) consist of metal particles with a size distribution on the sub-jim scale. Raman microscopy

388

M. Manfait and I. Nabiev

probes areas as small as 1 ixm^ on the surface. Hence, SERS-active surfaces must be area independent for SERS intensity enhancement, if any degree of reproducibility is to be obtained. The 'chemical' mechanism of enhancement (see Section LB) depends on the atomic scale roughness of the surface (<0.5 nm) - adatoms and clusters of adatoms (Otto et ai, 1992). These centers of adsorption are distributed evenly on the working area of a SERS-active substrate and, thus, the Raman enhancement due to a chemical mechanism must be area independent. As demonstrated by Van Duyne et al. (1986), the same is true for the electromagnetic contribution to SERS enhancement. The area independence of Raman enhancement due to the increase in the electromagnetic field near the sub-fxm defects of a metal surface might at first be unexpected. However, it is easily shown by an analysis of the SERS intensity expression for an infinitesimal area element on the surface (Van Duyne, 1979) that d3surf = A^surfi^(cla'/dn)<^)2e(a>s)-ir/L(r, e)rdrde,

(1)

where Ss^i-f is the detected intensity (photoelectron counts s~^) at the Stokes-shifted frequency o)^; Nsurf is the adsorbate density (molecules cm~^); n is the solid angle of the collection optical system (sr); and (dcr/dH) is the Raman scattering cross-section (cm^ molecule"^ sr~^), the value of which reflects any enhancement due to resonant interaction of the excitation frequency with states of the adsorbate/substrate system. In Eq. (1) {%)^ = L((o)^/L((o^)^ is the electromagnetic surface-averaged enhancement factor for the excitation and scattering frequencies; e(a)) is the energy of the incident laser photon (J); T is the product of the photodetector quantum efficiency, transmittance of the collection optics, and the throughput of the dispersion system; and /L(r, 6) is the laser beam irradiance (Wcm~^), which is in general a function of the polar coordinates. The quantity Ii^(r, 6) can always be written as a peak irradiance, incorporating the total incident power, PQ and the distance, w. It is characterized by the laser spot size multiplied by a normalized distribution function. For instance, the commonly used TEMQO Gaussian beam distribution is given by Siegman (1971) in the form (see Chapters 2 and 3)

Iir,e)=l^yM-2r'/w^).

(2)

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Here, w is the radius from the beam center at which the irradiance has fallen to 1/e^ of its peak value. By integrating Eq. (1) with a distribution function such as that given by Eq. (2), it is easily seen that 3surf is independent of w. Here it has been tacitly assumed that T and Ct are roughly equivalent in macroscopic and microscopic systems.

Applications in IVIedicine 389 III. RAMAN MICROSCOPY OF SINGLE LIVING CELLS AND CHROMOSOMES

As noted in Section II.A, the development of highly sensitive, confocal Raman instrumentation (Puppels et aL, 1990a) and the optimization of experimental parameters (Puppels et al., 1991) have ehminated the problem of degradation of biomedical samples. The Raman microspectrometer designed by J. Greve's group (Puppels et ai, 1990a,b) makes it possible to study single cells, chromosomes, etc. with a high spatial resolution ( < 1 juim).

Puppels et al, (1991) described a Raman microspectroscopic investigation of human granulocytes - cells with a dense granulation of the cytoplasm. Hence, the position of the nucleus (in the unstained cells) could be easily determined. Figure 4 shows spectra obtained from the cytoplasm (a) and the nucleus (b) of an intact human eosinophilic granulocyte. The spectrum of the nucleus consists of bands that can be assigned to DNA and protein vibrations which strongly resemble the reported spectra of isolated chromatin (Thomas et al., 1911 \ Savoie et al., 1985), as well as spectrum (c) of Fig. 4, which was obtained from a single metaphase chromosome. The cytoplasm spectrum differs dramatically from that of the nucleus. The amount of DNA present in the measured volume during the recording of the spectrum of the chromosome was estimated to be 50 fg, corresponding to 50 x 10^ base pairs. Puppels et al. (1990b) also recorded an excellent spectrum of metaphase chromosomes to obtain a better understanding of the origins of the banding patterns that appear after physicochemical treatment and staining. They measured the differences in the DNA/protein ratio between a band, an interband and a telomer of a fixed polytene chromosome. These authors investigated the possibihty that the differences are related to local variations in transcriptional activity by studying unfixed chromosomes as well. They also established the presence and induction of left-handed Z-DNA in such chromosomes. A wide range of application of the instrument developed in J. Greve's group can be expected in the analysis of small biological structures and, possibly, for monitoring biological events such as the cytotoxic process mediated by natural killer cells. The use of 660 nm laser light enables Raman measurements to be made on cells and chromosomes which have been labelled with fluorescein-conjugated antibodies, thus facilitating the prior localization of specific structures such as sites of transcription or replication by conventional fluorescence microscopy. Its combination with immunofluorescence techniques further increases the potential of the Raman microprobe in biomedical appHcations.

390

M. Manfait and I. Nabiev

1600

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wavenumbers ( c m - ^ ) Figure 4 Raman spectra of a single intact cell and a single metaphase chromosome. Spectrum (A) was obtained from a cytoplasmic region of a human eosinophilic granulocyte, spectrum (B) from the nucleus of the same cell and (C) from a chromatin of a metacentric Chinese hamster lung cell (V79) metaphase chromosome. The intensity scale is for (A); [for (B) it is multiplied by 5, for (C) by 1.25]. For clarity, the spectra have been shifted along the ordinate. Raman band assignments for the nucleus and chromosome spectra (B,C) are based on those by Thomas et al. (1977), Goodwin and Brahms (1978), Savoie et al. (1985), Tu (1986) and Otto (1987). The water and substrate spectra have been subtracted from spectra (A,B,C). Abbreviations: T, thymine; C, cytosine; A, adenine; G, guanine; RP, ribophosphate; BK, backbone; p, protein; a, a-helix; Tyr, tyrosine; Phe, phenylalanine; def, deformation; str, stretching. Experimental conditions: x63 Zeiss Plan, Neofluar water immersion objective (N.A. = 1.2); laser power 5mW (A,B), 10 mW (C); measuring times 150 s (A,B) or 900 s (C); granulocyte on poly-L-lysine-coated, fused-silica (fs) substrate in the standard PBS isolation method of Terstappen et al. (1986); chromosome on fs substrate in hypotonic buffer (4mmoir^ MgCl2, lOmmoll"^ Tris and lOmmoll"^ NaCl), isolation method of de Mul et al. (1984).

Applications in Medicine 391 IV. RAMAN MICROSCOPY OF OCULAR LENSES

The ocular lens is a good object to investigate nondestructively by means of laser Raman spectroscopy (Yu and Barron, 1986,1987,1988). The embryonic stage is at the nucleus (of the lens) center. So after an aging process, the oldest tissues are of the nucleus. The newly differentiated stage in the equatorial region. So, the youngest tissues are in the equator of the lens. Science-lens proteins are highly packed, especially in the nucleus center. They can yield high-quality Raman spectra in situ from the lens proteins. Yu and East (1975) reported the first Raman spectra of animal lenses. It was stated that a useful Raman spectrum can be recorded from a volume of 10"^ |xl (Yu et aL, 1985b), with only 1 mW of laser power and about 700-950 s of data integration time (Mathies and Yu, 1978). It would seem, therefore, to be a useful method for the investigation of macromolecular alterations in opaque cataractous spots. It is also considered to be a promising clinical diagnostic tool for senile cataracts (Mathies and Yu, 1978; Yu et aL, 1981-82). Raman spectra of intact human lenses have been recorded by several investigators (Kuck and Yu, 1978; Yu et aL, 1985a; Gijsbers et aL, 1986; Bot et aL, 1989). These authors were confronted with the following problems: (i) Excessive fluorescence from human lenses above the age of 20 years (Kuck and Yu, 1978; Yu and Barron, 1986; Yu et aL, 1985a). In order to eliminate the excessive fluorescence, a clear region in the intact lens was irradiated with laser Ught for many hours before recording a spectrum, and (ii) Difficulty in focusing precisely on the opaque and translucent regions. Yu et aL (1988) developed a fluorescence/Raman imaging system which scans the surface of a lens (Fig. 5). In this way the spectra are not affected by the presence of opaque regions in the body of the lens. This method offers a great advantage in cataract research. The fluorescence/Raman imaging system has a number of important capabilities, including multichannel detection of 'position-defined' fluorescence or Raman spectra from gridded points (1-8 jxm), scanning of micro- (10 jjim x 10 jxm) and macro(2.5 cm X 2.5 cm) samples, automated, simultaneous acquisition of intensity data of up to six spectral features (either peak or integrated intensities) from each point, and excellent stray light rejection, to allow detection of weak Raman bands from soUd samples, etc. To improve the accuracy in the position of the measurement and to avoid the problem of masking of the Raman signals by the strong fluorescence from human lenses over the age of 20-30 years, lens slices have been used (Bot etaL, 1989). Lens slices, even of very old human lenses, yield reUable Raman spectra, provided that they are pre-irradiated with laser light for several hours (Fig. 6). The Raman spectra obtained by this technique were qualitatively

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comparable to those obtained from young, nonfluorescent human lenses (Kuck and Yu, 1978; Yu et al., 1985a). The principal factor involved in the lower fluorescence of slices is due to the fact that less fluorescent material is excited in a slice. Consequently, the backscattering of fluorescent light into the objective from positions outside the measured volume will also be reduced. In addition the absorption of Raman and laser light above the focal spot is weaker in a slice.

SURFACE-ENHANCED RAMAN SCATTERING (SERS) MICROSCOPY

A. Substrates for SERS Microscopy of Biomedical Samples One of the major points in the application of SERS microspectroscopy is the preparation of metallic surfaces or media (so-called 'SERS-active substrates' or 'SERS-active surfaces') that have an easily controlled protrusion size and reproducible structures on the sub-|xm scale. The essential characteristics of substrates suitable for the SERS microscopy of biomedical samples have been described above (see Section II.B). The metal hydrosols, island films and nuclear pores coated by metals, among others, which have been prepared in this laboratory, have proved to be rather

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simple and inexpensive substrates having metal particle and size distribution uniformity on a sub-ixm scale that are particularly useful for the SERS microprobe. A number of researchers have attempted to develop methods to prepare metal substrates with control of particle shape and size. Two reviews of the relative merits of some of the substrates mentioned above have been published (Vo-Dinh et aL, 1988; Vo-Dinh, 1989). Attention will be focused here on the two types of sohd, SERS-active surfaces which have been used in biomedical appUcations of SERS: metal-island films (Schlegel and Cotton, 1991; Nabiev et aL, 1993; Sokolov et aL, 1993a) and nuclear pores, coated by the metal (Nabiev and Oleynikov, 1990; Oleynikov etaL, 1990), and liquid SERS-active substrates - metal hydrosols (for reviews, see Nabiev et aL, 1988, 1993a).

394

M. Manfait and I. Nabiev

Figure 7 Electron micrograph of a gold hydrosol prepared by HAUCI4 reduction with a solution of trisodium citrate. 1. Hydrosols Metal colloid hydrosols are often used to produce SERS-active media in solution. Important advantages of the SERS of colloidal systems are the simplicity of sample preparation and the ease of manipulation for analytical studies. Gold hydrosols containing particles of uniform size and regular spherical shape are usually prepared by reducing HAUCI4 with a solution of trisodium citrate. In so doing, a colloid consisting of gold spheres with diameters of about 20 nm (characterized by an absorption band in the 500-550 nm region) is formed. By varying the relative concentrations of the metal salt and the

Applications in Medicine

1600

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Figure 8 Micro-SERS spectrum of doxorubicin recorded under the microscope with the laser beam focused at a diameter of c. 0.5 ixm on the hydrosol coagulate. A water immersion objective (50x) was used with a laser power (at 514.5 nm) of 30 |xW on the sample.

reducing agent, it is possible to control the size of the metal particles, depending on the purpose of the experiment (Fig. 7). Silver hydrosols for SERS are usually prepared by reducing silver nitrate with trisodium citrate (Nabiev et al., 1988, 1993) or with sodium borohydride (Nabiev et aL, 1988). They are very stable (for many weeks) because each particle is charged. Metal particles are usually negatively charged because of the adsorption of anions. When a neutral molecule is added to the metal hydrosol, it can adsorb; the neutral molecule replaces an adsorbed anion, reducing the charge of the particle and increasing the possibility of particle coagulation. This process depends on the size and shape of the hydrosol particle, the ionic strength, the pH, and the adsorbate concentration. Transmission electron microscopy is employed to monitor the sizes and shapes of the colloidal particles.

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of the spheres of c. 30 nm, and an average length of the rods of c. 60 nm. These dimensions were determined by transmission electron microscopy. The density of the nonaggregated silver particles was approximately 3.2 x 10^^ 1~^ (Hildebrandt and Stockburger, 1984), and the concentration of doxorubicin in the colloidal suspension was 10~^moll~^. Simple calculations made as described by Hildebrandt and Stockburger (1984) show that the micro-SERS spectrum presented in Fig. 8 was recorded from approximately 500 molecules of doxorubicin. This extra-high sensitivity of the SERS-microprobe technique offers the possibility of detecting the spectrum of the drug in treated, single living cells. Another example of hydrosol applications of SERS microprobe spectroscopy is shown in Fig. 10. The SERS spectrum of intoplicine, a new antitumor drug recently proposed for clinical tests (Fig. 9), was recorded by focusing

398

M. Manfait and I. Nabiev

Applications

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the laser beam on one coagulate of the silver micelles with a diameter of the order of 1 ixm. The spectrum was obtained from less than 600 molecules of intoplicine. This compound has a quite low quantum yield of fluorescence as compared with that of doxorubicin. Thus, it was possible to record its normal Raman spectrum in the macrochamber. Figure 10 shows that the micro-SERS and the normal Raman spectrum of intoplicine are quite similar. However, even the bulk concentration at which intoplicine was introduced in the hydrosol is six orders of magnitude less than that used to obtain the normal Raman spectrum. The SERS microprobe analysis of intoplicine distribution inside a living cell will be presented in Section V.B.2. 2. Vacuum-deposited Metal Island Films Vacuum-deposited, silver island films display extinction bands in the visible and near-IR regions (Fig. 11). These bands correspond to the excitation of the transverse, collective electron resonances of the metal particles. The wavelength maxima and half-widths of the absorption bands depend strongly on the thickness of the film, and the rate of silver deposition (Schlegel and Cotton, 1991; Sokolov et al, 1993a). The greatest Raman enhancement is produced by the SERS-active island films with extinction maxima near 520 nm (Sokolov et al., 1993a). For these films the distance between silver particles does not exceed their size (Fig. 11). For this reason interparticle interaction gives rise to an increase in the local field near the metal surface. For island films with extinction maxima at shorter wavelengths this effect is less important because the distance between particles is greater. For island films with extinction maxima at wavelengths longer than 520 nm, the distance between islands corresponds to direct contact between the metal particles. The result is a decrease in the resonance-enhancement properties of the islands and, hence, a decrease in the SERS intensity. The SERS microprobe spectrum of j8-carotene (Fig. 9) adsorbed on a silver island film is shown in Fig. 12. The SERS spectrum of j8-carotene adsorbed directly on the surface of a silver island film differs from its resonance Raman spectrum in the upshift of the bands of the adsorbed species. Two monolayers of stearic acid were deposited on a silver island film by standard LangmuirBlodgett transfer techniques. The difference between the SERS and the resonance Raman spectra of j8-carotene vanishes when the j8-carotene molecules are separated from the silver surface by two monolayers of stearic acid molecules (Fig. 12). Only a slight decrease in the signal intensity (c. 30%) is detected for j8-carotene molecules removed from the surface by a distance of approximately 5 nm - the thickness of a stearic acid bimolecular Figure 11 Electron micrographs (A-C) and absorption spectra (D) of silver island films of different thicknesses, prepared by thermal evaporation of silver on glass slides. Silver mass thicknesses: 4nm (A), 8nm (B) and 14nm (C).

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Figure 12 Resonance Raman (a) and micro-SERS (b,c) spectra of j8-carotene adsorbed on a silver island film, (b) Sample directly in contact with the silver surface, and (c) spaced from the metal surface by a distance of approximately 5 nm by deposition of two monolayers of stearic acid on a silver island film. stack. This observation suggests that there is no appreciable contribution of a short-range enhancement mechanism to the SERS of j8-carotene on the silver island film. The observed differences between SERS and resonance Raman spectra can be attributed to electrostatic interactions of the conjugated C = C system with the metal, rather than to short-range, chemical interactions. The SERS-microprobe analysis of the intracellular distribution of this type of molecule on the silver island films is presented below. Gold island films (Fig. 13) have been employed in SERS applications.

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Figure 13 Electron micrographs of gold island films of different thicknesses, prepared by thermal evaporation of gold on glass sHdes: (a) 8 nm, (b) 14 nm.

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LAYER OF METAL DEPOSITED BY GALVANOMETRICAL TECHNIQUE

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Figure 14 The principal steps in the preparation of nuclear-pore, SERS-active substrates. These substrates have absorption bands in the red and near-IR regions and could be used effectively in experiments with red excitation and in FT-SERS. 3. Nuclear Pores Another type of solid, SERS-active substrate is a nuclear-pore filter coated with silver. This SERS-active substrate was first produced and patented by one of us (Nabiev and Oleynikov, 1990; Oleynikov et al, 1990). The process consists of etching the support with ions of a given density and type, chemical treatment of the etched support, and deposition of a metallic layer at a

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specific angle with simultaneous spinning of the support. These operations are followed by metallization of the surface and removal of the support (Fig. 14). The required surface roughness is obtained by etching the support material, which must be an insulator (e.g. polyethyleneterephtalate, a polycarbonate, a polyimide, polypropylene, mica, quartz or glass) with high-energy ions (1-10 MeV per nucleon), which are produced by the accelerators such as the U-300, U-400 or IC-100 (United Institute of Nuclear Research, Dubna, Russia). A flux of high-energy ions produces latent tracks in the support material, as shown in Fig. 14. Etching of a polymeric support material is made with an alkaH solution. Cylindrical pores or conical holes are produced in this step. The shape of these pores or holes depends on the type of etching ions, their masses and energies, and the conditions of the process of track development temperature and chemical concentration. Finally, these etched and developed supports are coated with a metallic layer which is obtained by thermal evaporation. The thickness of the metallized coating must be sufficient to fill the holes or to create sharp edges inside the pores. A metallic deposition that is too thick can close the pores, while a deposition that is too thin will not create sharp edges. Both of these cases decrease the SERS effect. The optimal thickness of the metal layer has been determined to be in the range 10-200 nm. The angle of direction of the silver evaporation was chosen to be 45° with respect to the axis of the pores so that sharp edges were formed inside the pores and the holes were completely filled with the metal. Both substrates produced by this method (see Fig. 15) are very effective in SERS appUcations. The sample under investigation can be deposited on the 'SERS-active' surface by simply dropping the sample solution onto the substrate. These substrates provided very strong (by a factor of up to 10^) electromagnetic enhancement of the Raman cross-section (Nabiev and Oleynikov, 1990; Oleynikov et «/., 1990). B. SERS Microscopy of Living Cells and Drug Pharmacokinetics

The utiUty of the SERS microprobe technique will be illustrated by studies of intact biological organisms, living cancer cells and living cancer cells treated by drugs. Electrochemically micromachined Ag tips with diameters on the order of 1-5 juim, silver island films and silver hydrosols were used as SERS-active substrates in these experiments. 1. SERS Microprobe Analysis of Intact Biological Organisms Recently Todd and Morris (1993) demonstrated a simple method for electrochemically micromachining SERS electrode probes. The Ag tips were

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•'•^\

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Figure 15 Electron micrograph of the surfaces of SERS-active substrates prepared by the procedures shown in Fig. 14. electrochemically micromachined by oxidizing the Ag at a positive potential to produce tip diameters on the order of 1-5 jxm. The SERS spectra of model compounds (pyridine and histamine) on these microtips were easily detectable, demonstrating that, although the SERS-active surface was three orders of magnitude smaller than that which had been used previously, a 2 mm diameter rough, silver electrode, there was no qualitative difference in the resulting SERS spectra. Another interesting experiment was carried out in order to demonstrate the feasibility of positioning the probe in a restricted volume and to check if the volume constriction could affect the excitation or collection of the SERS signal. To achieve this goal, Todd and Morris (1994) used nuclear pores (see Section V. A.3). The diameter of the randomly distributed pores was c. 10 |xm and the depth approximately 6 ixm. Hence, the internal pore volume was about 500 fl. The filter was placed in a droplet of methyl orange solution and the SERS spectra were obtained with a 2 |xm diameter electrode tip inside one of the pores of the filter. The resulting spectra were compared with the control spectra, which were obtained without the membrane present. Well-resolved spectra collected in both configurations showed that the constricting volume did not affect the excitation or collection of the SERS signal. Finally, Todd and Morris (1994) performed a functional test of Ag microprobes operating through a membrane and in a complex biological matrix. This experiment was made with the use of fertilized zebrafish eggs, which are 2 mm diameter spheroids. The eggs consist of the outer chorion (mixture of proteins), the yolk (mixture of carotenoid pigments) and the

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embryo (complex matrix containing small peptides and nucleotides). Todd and Morris (1994) were able to insert the Ag microprobe into either the yolk or the embryo cells without damage (Fig. 16a) and to record corresponding SERS spectra from the different parts of the egg. The SERS spectra of the yolk were dominated by the characteristic modes of the Raman carotenoid bands (Fig. 16b), whereas the embryo spectra contained mainly characteristic bands of the embryo proteins and nucleosides. The experiments described above demonstrated: (i) That the Raman spectra obtained with ixm-scale Ag- microprobes were the same as those of their macroscopic counterparts, (ii) The feasibility of precise positioning of these small probes in restricted volumes on the order of 500 fl and, finally, (iii) The detection of high-quahty SERS spectra from the different compartments of the intact biological organism. Todd and Morris (1994) also proposed to use their probes as real-time detectors in a flow system such as an effluent stream from a microdialysis probe and in capillary electrophoresis. 2. SERS Microprobe Studies of Dimethylcrocetine and Ellipticine in Single Living Cells on Silver Island Films and Hydro sols As shown by Sokolov et al. (1993a), for a silver island film the short-range mechanism of Raman enhancement contributes primarily to enhanced Raman scattering from the molecules with electronic transitions in the UV region or with low extinction coefficients in the visible region. On the other hand the long-range, classical electromagnetic mechanism predominates in the spectroscopic enhancement of molecules with high extinction coefficients in the visible region. The latter mechanism is applicable to studies of dimethylcrocetine (DMCR) - a lipophilic derivative of crocin (the principal coloring component of the extract from the natural pigment of Crocus sativus) - which has been recognized as an agent of cell differentiation. The DMCR chromophore (Fig. 10) has a very high extinction coefficient for the electronic transition in the visible region. Thus, the long-range mechanism contributes mainly to the Raman enhancement of this molecule when it is deposited near the surface of a silver island film (Sokolov et al., 1993a). Hence, it is reasonable to use silver island films for studies of DMCR inside living cells. After treatment of K562 and Friend cells, they are deposited on the silver island films which have been previously treated by DMCR. The spectrum of the pigment can be detected in the nucleus, as well as that of the pigment in the cytoplasm of the cells (Fig. 17). The spectra demonstrate the accumulation of DMCR in the cytoplasm of the Friend cells, but an absence of this drug in their nuclei. Moreover, new components at c. 1280 and

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Figure 16 (a) Video microscope image of 1.5-h-old zebrafish egg showing structure and the probe tip inserted in an embryo cell, (b) SERS spectra of the yolk of a zebrafish egg acquired with a 3 jxm diameter Ag probe tip with 400 |JLW for 10 s.

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Wavenumber Ccm*^) Figure 17 Micro-SERS spectra of free dimethylcrocetine (DMCR): (a) DMCR in the cytoplasm of K562, (b) in the cytoplasm of Friend cells, (c) in the K562 nucleus, (d) the resonance Raman spectrum of K562 cells treated with DMCR deposited on a glass slide and recorded under the microscope (e). The laser power was 10 |JLW at a wavelength of 514.5 nm (Manfait et al., 1991). 1150 cm~^ appear in the spectrum of the intracellular pigment, as compared with that of free DMCR. For the K562 strain the spectra of DMCR located in the nucleus or in the cytoplasm are of comparable intensity. Thus, the SERS spectra reveal differences in the interaction of this drug with the diverse cell strains, as well as to provide an evaluation of its penetration inside the nucleus or the cytoplasm. A liquid SERS-active substrate - silver hydrosol - has been used for the SERS-microprobe analysis of the interaction of eUipticine (a natural plant alkaloid which displays some antitumor properties) with living cancer K562 cells. These molecules have a planar, conjugated, polycyclic aromatic chromophore and interact with DNA by intercalation between base pairs (Liu, 1989). The apparent pK of eUipticine in vitro is strongly effected when this weak base is bound to its target: pK = 7.4 in aqueous solution; pK = 9 in bound form with DNA. In the cell, eUipticine may exist in both the cationic, protonated form and in the neutral form. A silver hydrosol prepared by the

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Figure 18 Micro-SERS spectra of ellipticine (3 |xm): (a) pH8; (b) pH5.5; (c) complexed with DNA, pH 8. Micro-SERS spectra of K562 cancer cells treated by 10 |xmol 1~^ of ellipticine recorded by focusing a laser beam on the nuclei (N^ and N2) and on the cytoplasm (Cj and C2) of two cells. The laser power on the sample was 20jxW at 514.5 nm (Millot et «/., 1991). reduction of AgN03 with trisodium citrate was aggregated by sodium perchlorate before introducing K562 cancer cells treated by ellipticine. The cell pellet was incubated in the aggregated hydrosol and the micro-SERS spectra were recorded (Fig. 18) with the use of an Omars 89 (DILOR) micro-Raman spectrometer equipped with an Olympus BH-2 microscope (lOOx water immersion objective). The intensities and shapes of the main bands at 1588 and 1406 cm~^ in the SERS spectra of ellipticine proved to be very sensitive to the protonation state of the molecule. The value of the intensity ratio ^588^406 is greater than that observed when the ellipticine is in the cationic form (Fig. 18B) and is less than that obtained when it is in the neutral form (Fig. 18A). In the presence of DNA, the ^K value of ellipticine in the bound form shifts from 7.4 to 9. Curve (c) of Fig. 18 corresponds to spectrum b of the protonated form of ellipticine. In the micro-SERS spectrum of K562 cells treated by ellipticine, the ratio ^588/^406 is greater than that measured when the laser beam is focused on the nuclei.

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the nuclei. This result suggests that in the nucleus the drug is intercalated inside the DNA double helix in its cationic form, as expected from the ^K of ellipticine bound to DNA in vitro. In the SERS spectra recorded by focusing the laser beam on the cytoplasm, the value of 3i588/3i406 is also greater than 1. However, the intensity of the 1406 cm"" ^ band is higher, as compared with that observed in the intranuclear spectra. This result could arise from a contribution of neutral ellipticine molecules within a cytoplasmic compartment such as mitochondria. This interpretation has been suggested before on the basis of micro-spectrofiuorimetric data. 3. Doxorubicin Pftarmacolcinetics Probed by SERS Microspectroscopy of a Living Cell The SERS microprobe approach has been appUed to the analysis of the intracellular distribution of doxorubicin (Fig. 10), an antitumor drug which is systematically used for clinical trials. Conventional resonance Raman spectra of doxorubicin can only be recorded at concentrations greater than lO^'^mol \~^ and are accompanied by a very strong fluorescence background (Manfait et al., 1982). The SERS spectrum of this drug is similar to its resonance Raman spectrum (Manfait et al., 1982) and is characteristic of the chromophoric parts of the molecule (Fig. 9). The fluorescence of doxorubicin adsorbed on a silver hydrosol is totally quenched. The ultrahigh sensitivity of SERS for this kind of molecule facihtates the measurement of spectra at concentrations down to 10~^^mol 1~^ and enables the development of a rapid method of detecting antitumor drugs in living cells (Nabiev et al., 1991b). For the micro-SERS studies of cancer cells treated by doxorubicin, three cell preparations have been employed, namely: (i) The cells were introduced into the hydrosol, incubated there and washed to remove the hydrosol external to the cells before the Raman measurements. (ii) The cells were incubated with doxorubicin, washed to remove the doxorubicin outside the cell and then introduced into the hydrosol in the same manner as in the first preparation. (iii) The cells were introduced into a hydrosol which already contained doxorubicin, incubated there, and washed to remove the hydrosol, as well as the hydrosol-doxorubicin complexes outside the cells. The objective of the initial procedure (i) was to introduce hydrosol micelles inside the living cells via endocytosis. This preparation will be referred to as 'untreated ceUs'. The hydrosol was introduced into the cell, as indicated by image analysis; the analyzer showed that the silver hydrosol was actually incorporated into the living cells (Fig. 19). The viability of cells incubated

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Figure 19 Digitized images of K562 cells: (A) control, (B) samples incubated in the presence of silver hydrosol. Endocytosis is evident in (B) as compared with the control (A) (Nabiev et aL, 1991b).

with silver hydrosol was checked by microscopy with Trypan blue, and the percentage survival was always greater than 95% (Nabiev et al., 1991b). Procedure (ii) led to the accumulation of doxorubicin in the nuclei of the cells (Manfait et aL, 1982). This preparation will be described by 'living cells with DOX in the nuclei' or 'living cells treated by DOX'. Procedure (iii) was used to incorporate doxorubicin-hydrosol complexes into the cytoplasm through endocytosis. This preparation is called 'living cells with doxorubicin in the cytoplasm' (Nabiev et al., 1991b). The striking feature of the micro-SERS spectrum of 'untreated cells' when the laser beam is focused on the cell nucleus is the band at approximately 731 cm~^. This feature is characteristic of the adenine ring-breathing vibration (see Fig. 20). This band is not seen in the Raman spectra of living cells which have not been incubated with silver hydrosol, nor in the micro-SERS spectra of 'untreated cells' when the laser beam is focused on the cytoplasm. The hydrosol used is more specific to adenine than to other nucleotides because it was activated by chloride ions contained in the PBS (Nabiev et aL, 1990). The SERS spectrum of living cells with doxorubicin in the nuclei is very different from the spectra of cells with doxorubicin in the cytoplasm, but is closely correlated with that of the in vitro complex of doxorubicin with calf thymus DNA (Fig. 20). The main spectral differences associated with doxorubicin-(target in nuclei) complex formation manifest themselves in

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Figure 20 Micro-SERS spectra of K562 cells treated by doxorubicin, recorded under the microscope at the focus of the laser beam on (a) the cytoplasm or (b) the nucleus of the cell and (c) the SERS spectrum of a complex of doxorubicin with calf thymus DNA (one molecule of doxorubicin per 1000 base pairs in vitro; Nabiev et al., 1991).

changes of the relative intensities of the bands in the 1200-1255 cm~^ region and at 1450 cm~^, as well as an increase in the intensity of the band near 1630 cm~^. These results are well correlated with the spectral differences initiated by the doxorubicin-DNA complex formation in vitro. The spectrum of doxorubicin in the cytoplasm is also different from the spectrum of free doxorubicin adsorbed on a hydrosol (Fig. 20). This observation impHes that doxorubicin in the cytoplasm has a target which is somewhat different from that (DNA) in the cell nucleus. 4. Micro-SERS Analysis of Intoplicine in a Living Cell and In Vitro Modelling of its Intracellular Interactions One of the most recent and important discoveries in the field of cancer research has been the identification of DNA topoisomerases as targets for several classes of antitumor drug (Liu, 1989; Wang, 1989). DNA

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topoisomerases (Topo I and Topo II) are nuclear enzymes that interconvert topological isomers of DNA by breaking and releasing phosphodiester bonds. They are thus involved in transcription, replication, chromosome segregation and DNA repair (Wang, 1989). A number of anticancer drugs specifically inhibit Topo II (D'Agra and Liu, 1989). By stabilizing an intermediary complex of the Topo II reaction, these drugs block the relegation of breaks in the DNA strand. Stabilization of the Topo II-DNA complex is thought to interfere with replication and transcription in rapidly growing cancer cells and to lead ultimately to cell death (Liu, 1989; D'Agra and Liu, 1989). Intoplicine (RP 60475, NSC 645008; Fig. 10) is representative of a new series of topoisomerases inhibitors (Nguyen etal., 1990). This compound has been selected for cUnical tests (Nguyen et al., 1990, 1992) because it displays potent activities in various cellular and animal models and it inhibits topoisomerases I and II (Bissery et al., 1990; Riou et al., 1992; Poddevin et aL, 1993). The micro-SERS spectrum of intoplicine in the living cancer cell K562 was recorded and compared with those of its complexes with DNA and Topo II in vitro (Morjani et al., 1993). After incubation of the K562 cells with 1 ixmol 1~^ of intoplicine for 1 h and with silver hydrosol for 30 min at 37°C, the micro-SERS analysis was performed on a single living cell. Figure 21 shows spectral differences in the range 1360-1416 cm~^ between intoplicine situated in the nucleus and in the cytoplasm [compare curves (b) and (c)]. These spectral differences correspond to those observed in vitro in the relative intensities of the 1360-1416 cm~^ band for the drug in the free form and in the ternary complex [compare curves (a) and (d) of Fig. 21]. These results suggest that within cells, intopHcine interacts with Topo II in the nucleus, whereas it remains in the free form in the cytoplasm. The detailed analysis of the spectral differences found in vitro and their comparison with the results obtained from living cells has been presented elsewhere (Nabiev et al., 1994c).

5. SERS Microprobe Detection of Sialic Residues on the Membrane of a Living Cell As noted before, the advantages of SERS spectroscopy include not only enormous enhancement of the Raman cross-section of molecules in the vicinity of a metal surface, but also the possibility of studying the topology of complex systems (e.g. membrane-bound proteins and cells) due to the short-range character of the Raman enhancement in certain SERS-active media (Nabiev et «/., 1993). The position of the sialic acid (SA) residue as a terminal sugar of the oHgosaccharide chains of glycoproteins and glycolipids (Schauer, 1982) makes it accessible to the metal surface in SERS-active systems. Sialic acid residues

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1600 .1400, 1200 wavenumber ( c m - l ) Figure 21 SERS spectra of intoplicine (a) in the free form, (d) in the ternary complex with DNA and Topo II, (b) in the cytoplasm and (c) in the nucleus of K562, a living cancer cell. Experimental conditions for (a) and (d) are as in Fig. 10. Experimental conditions for (b) and (c): laser power 20 jxW; 100 accumulations (2 s each); lOOx water immersion objective was used. mediate a variety of biological processes. Sialyloligosaccharides serve as receptor determinants for influenza and other viruses (Wiley and Skehel, 1987). Oncotransformation and some somatic diseases are accompanied by distinct changes in sialylated glycoconjugates (Schauer and Yamakawa, 1988).

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1000 Raman Shift (cm''') Figure 22 SERS spectra of the ai-acid glycoprotein isolated from (a) a healthy donor (n-AGP), and (b) from a tumor patient (c-AGP). The concentrations were approximately 10~^moll~^ for the glycoproteins. The laser power on the sample was 10 mW at a wavelength of 514.5 nm. The spectra were recorded with the sample in the macrocell.

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The SERS spectra (Fig. 22) have been recorded of two human sialyl-aiacid glycoproteins isolated from a blood serum of 20 healthy donors (n-AGP), and from an ascitic fluid of four stomach cancer patients (c-AGP). After removal of the SA residues from the sugar chains, n-AGP and c-AGP yielded virtually identical spectra (Sokolov et al., 1993b), although with dramatically decreased band intensities (Fig. 22). It has been shown that irreversible conformational changes of AGP molecules occur on desialylation of proteins by heating or on acidic hydrolysis (Vakurina, 1992), which is not the case in neuraminidase treatment (Tripodi et al., 1971). Sialic acid residues were removed either by neuraminidase treatment or by acidic hydrolysis. In both cases an analogous decrease in SERS spectral intensities was observed. These data indicate that for the SERS-active media employed, the asialo part of the glycoproteins makes a negligible contribution to the SERS spectra, which are characterized by the vibrational bands of the SA residues. The normal and cancer AGP differ in SA content. The higher SERS intensity for the cancer AGP, as compared with the normal AGP, correlates well with the different SA content in these two glycoproteins (Sokolov et al., 1992, 1993b). These results demonstrate that SERS spectroscopy can be used to detect SA residues and changes in their organization in the membrane glycoproteins at very low concentrations. Hence, it was reasonable to apply the microSERS method for the detection of SA residues on the membrane of a living cell. The same technique was applied previously to study the distribution of doxorubicin and intoplicine inside the cell (see Sections 2 and 3 above). The population of myeloma X-63 cells was incubated with the aggregated silver hydrosol. Intense Raman spectra were obtained from the SA residues under the microscope after focusing the laser beam in the region of the cell membrane (Fig. 23). These spectra were more intense than those of doxorubicine or intoplicine due to the strong adsorption by the hydrosol micelles on the cell membrane. The SERS spectrum of the cell membrane is determined by the SA residues; the band positions correlate well with the SERS spectrum of a sialylated, ai-acid glycoprotein membrane (see Fig. 23). Hence, the SERS microprobe can be used as a rapid detection and conformational analysis technique for SA residues in a cell membrane, affording earlier diagnosis of some diseases, including cancer.

VI. SUMMARY

In principle, Raman microscopy can be used extensively in the study of biomedical samples, including biological tissues, intact organisms, cells and chromosomes. However, the inherently low efficiency of conventional Raman

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Figure 23 SERS spectra of (a) aj-acid glycoprotein isolated from a tumor patient (c-AGP), and (b) of a single X-63 cell. Spectrum (a) was recorded with the sample in the macrocell; the experimental conditions were as in Fig. 22. Spectrum (b) was recorded with the sample under the microscope (50x water immersion objective) at the focus of the laser light in the region of the cell membrane; the laser power on the sample was 50jjiW at 514.5 nm.

scattering and the risk of sample damage have been the main obstacles to the development of the Raman microprobe as a practical biomedical technique. The problem can be solved in two ways. One is to improve the sensitivity of the instrumentation, and the other is to enhance the Raman signal. Both of these complementary methods have been realized. Recent advances in the development of highly sensitive, confocal Raman microspectrometers, as well as the application of the SERS-microprobe

Applications in IVIedicine 417 technique, have created new horizons in the field of biomedical apphcations. It is clear from the results presented in this review that the use of micro-Raman spectroscopy as a practical biomedical technique is now a reality. With further improvements in both SERS-active substrate preparation and in instrumentation, Raman microscopy will not only complement the other biochemical methods, but will also ofifer the attractive features of superior mass sensitivity, molecular specificity and in situ applicability.

ACKNOWLEDGEMENTS The contributions to this field by the authors' laboratory were made possible by the efforts of a number of collaborators and colleagues. It is a pleasure to express our thanks.to H. Morjani, J.-M. Millot, J. F. Angiboust, and S. Sharonov (University of Reims, France) and A. Feofanov, K. Sokolov, V. Oleynikov and I. Kudelina (Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow). We also wish to acknowledge very useful conversations concerning different parts of this work with T. M. Cotton, M. D. Morris, G. Chumanov, J. Greve, S. Asher, P. Hildebrandt and R. E. Hester.

REFERENCES Ashkin, A. and Dziedzic, J. M. (1987). Science 235, 1517-1520. Bissery, M. C , Nguyen, C. H., Bisani, E. and Lavelle, F. (1990). Proc. Am. Assoc. Cancer Res. 31, 2747. Bot, A. C. C , Huizinga, A., De Mul, F. F. M., Vrensen, G. F. J. M. and Greve, J. (1989). Exp. Eye Res. 49, 161-169. Boyd, C. T., Rasing, T., Leit, J. R. R. and Shen, Y. R. (1984). Phys. Rev. B 30, 519-526. Brakenhoff, G. J., Blom, P. and Barends, P. (1979). /. Microsc. Ill, 219-232. Calmettes, P. P. and Berns, M. W. (1983). Proc. Natl. Acad. Set. USA 80, 7197-7199. Chang, R. K. and Furtak, T. E. (eds) (1982). Surface-Enhanced Raman Scattering. Plenum Press, New York & London. Coohill, T. P., Peak, M. J. and Peak, J. G. (1987). Photochem. Photobiol. 46, 1043-1050. DAgra, P. and Liu, L. F. (1989). Biochim. Biophys. Acta 989, 163-177. Delhaye, M. and Dhamelincourt, P. (1975). /. Raman Spectrosc. 3, 33^3. de Mul, F. F. M., van Welie, A. G. M., Otto, C , Mud, J. and Greve, J. (1984). /. Raman Spectrosc. 15, 268-272. Feofanov, A., Sharonov, S., Valisa, P., Da Silva, E., Nabiev, I. and Manfait, M. (1995). Rev. Sci. Instrum. 66, 1-13.

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Gijsbers, G., Vrensen, G., Willekens, B., Maatman, D., De Mul, F. and Greve, J. (1986). In: J. Stepanek, P. Anzenbacher, and B. Sedlacek, (eds), Laser Scattering Spectroscopy of Biological Objects. Elsevier, Amsterdam, pp. 583-594. Goodwin, D. C. and Brahms, J. (1978). Nucl Acid Res. 5, 835-850. Hildebrandt, P. and Stockburger, M. (1984). /. Phys. Chem. 88, 5935-5948. Kuck, J. F. F. and Yu, N.-T. (1978). Exp. Eye Res. 27, 737-741. Liu, L. F. (1989) Ann. Rev. Biochem. 58, 351-375. Manfait, M., Morjani, H., Millot, J.-M., Debal, V., Angiboust, J.-F. and Nabiev, I. R. (1990). In: N. I. Koroteev and M. Poroshina (eds), Laser Applications in Life Sciences, Part U: Lasers in Biophysics and Biomedicine. SPIE Proceeding Series, Washington, DC, pp. 695-707. Manfait, M., Morjani, H., Efremov, R., Angiboust, J.-F., PoHssiou, M. and Nabiev, I. (1991). In: R. E. Hester and R. B. Girling (eds). Spectroscopy of Biological Molecules. Royal Society of Chemistry, Cambridge, pp. 303-304. Manfait, M., Riou, J.-F., Morjani, H., Lavelle, F. and Nabiev, I. R. (1992a). In: W. Kiefer, M. Cardona, G. Schaack, F. W. Schneider and H. W. Schrotter (eds), Raman Spectroscopy. John Wiley & Sons, New York, pp. 520-521. Manfait, M., Nabiev, I. and Morjani, H. (1992b). /. Cell Pharmacol. 3, 120-125. Mathies, R. and Yu, N.-T. (1978). /. Raman Spectrosc. 7, 349-352. Millot, J.-M., Morjani, H., Aubard, J., Pantigny, J., Nabiev, I. and Manfait, M. (1991). In: R. E. Hester and R. B. GirUng (eds). Spectroscopy of Biological Molecules. Royal Society of Chemistry, Cambridge, pp. 305-306. Morjani, H., Riou, J.-F., Nabiev, I., Lavelle, F. and Manfait, M. (1993). Cancer Res. 53, 4784^790. Nabiev, I. R. and Manfait, M. (1992). In: W. Kiefer, M. Cardona, G. Schaack, F. W. Schneider and H. W. Schrotter (eds), Raman Spectroscopy. John Wiley & Sons, New York, pp. 666-667. Nabiev, I. and Manfait, M. (1993). Revue de I'lnstitut Frangais du Petrole 48, 261-285. Nabiev, I. R. and Oleynikov, V. A. (1990). The Technique of Identification of Structure and Ingredients of Materia. USSR Patent no. 4683200/25-603382. Nabiev, I. R., Efremov, R. G. and Chumanov, G. D. (1988). Sov. Phys. Usp. 31, 241-262. Nabiev, I. R., Sokolov, K. V. and Voloshin, O. N. (1990). J. Raman Spectrosc. 21, 333-337. Nabiev, I., Sokolov, K., Morjani, H. and Manfait, M. (1991a). In: R. H. Hester and R. B. Girling (eds). Spectroscopy of Biological Molecules. Royal Society of Chemistry, Cambridge, pp. 345-348. Nabiev, I. R., Morjani, H. and Manfait, M. (1991b). Eur. Biophys. J. 19, 311-316. Nabiev, I. R., Sokolov, K. V. and Manfait, M. (1993). In: R. J. H. Clark and R. E. Hester (eds), Biomolecular Spectroscopy. John Wiley & Sons, Chichester, pp. 267-338. Nabiev, I., Chourpa, I. and Manfait, M. (1994a). /. Phys. Chem. 98, 1344-1350. Nabiev, I., Chourpa, I. and Manfait, M. (1994b). /. Raman Spectrosc. 25, 13-23. Nabiev, I., Chourpa, I., Riou, J.-F., Nguyen, C. H., Lavelle, F. and Manfait, M. (1994c). Biochemistry 33, 9013-9023. Nabiev, I., Baranov, A., Chourpa, I., Beljebbar, A., Sockalingum, G. D. and Manfait, M. (1995). J. Phys. Chem. 99, 1608-1613. Nguyen, C. H., Lhoste, J. M., Lavelle, F., Bissery, M. C. and Bisagni, E. (1990). /. Med. Chem. 33, 1519-1528. Nguyen, C. H., Lavelle, F., Riou, J. F., Bissery, M. C , Huel, C. and Bisagni, E.

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(1992). Anti-Cancer Drug Design 7, 239-251. Oleynikov, V. A., Sokolov, K. V., Hodorchenko, P. V. and Nabiev, I. R. (1990). In: S. A. Akhmanov and M. Poroshina (eds), Laser Applications in Life Sciences. Part /.• Laser Diagnostics of Biological Molecules and Living Cells - Linear and Nonlinear Methods. SPIE Proceeding Series, Washington, DC, pp. 164-166. Otto, C. (1987). Ph.D. thesis. University of Twente, Enschede (Netherlands). Otto, A., Mrozek, I., Grabhorn, H. and Akermann, W. (1992). /. Phys. Condens. Matter 4, 1143-1212. Poddevin, B., Riou, J. R , Lavelle, F. and Pommier, Y. (1993). Mol. Pharmacol. 44, 767-774. Puppels, G. J., Huizinga, A., Krabbe, H. W., de Boer, H. A., Gijsbers, G. and de Mul, F. F. M. (1990a). Rev. Sci. Instrum. 61, 3709-3712. Puppels, G. J., de Mul, F. F. M., Otto, C , Greve, J., Robert-Nicoud, M., Arndt-Jovin, D. J. and Jovin, T. M. (1990b). Nature 347, 301-303. Puppels, G. J., Olminkhof, J. H. F., Sergers-Nolten, G. M. J., Otto, € . , de Mul, F. F. M. and Greve, J. (1991). Exp. Cell Res. 195, 361-367. Riou, J. F., Foss, I., Bissery, M. C., Larsen, A., Nguyen, C. M., Grondard, L., Saucier, J. M., Bisagni, E. and Lavelle, F. (1992). Proc. Am. Assoc. Cancer Res. 33, 2611. Rosasco, G. J., Etz, E. S. and Cassatt, W. A. (1975). Appl. Spectrosc. 29, 396-404. Savoie, R., Jutier, J.-J., Alex, S., Nadeau, P. and Lewis, P. N. (1985). Biophys. J. 47, 451-459. Schauer, R. (1982). Adv. Carbohydr. Chem. Biochem. 40, 131-139. Schauer, R. and Yamakawa, T. (eds) (1988). Sialic Acids. Kieler-Verlag Wissenschaft + Bindung, Berlin. Schlegel, V. L. and Cotton, T. M. (1991). Anal. Chem. 63, 241-247. Sharonov, S., Chourpa, L, Morjani, H., Nabiev, I. and Manfait, M. (1994a). Anal. Chim. Acta 290, 40-47. Sharonov, S., Nabiev, I., Chourpa, I., Feofanov, A., Valisa, P. and Manfait, M. (1994b). / . Raman Spectrosc. 25, 699-707. Siegman, A. E. (1971). Introduction to Lasers and Masers. McGraw-Hill, New York, ch. 8. Smulevich, G. and Feis, A. (1986). / . Phys. Chem. 90, 6388-6392. Smulevich, G. and Spiro, T. G. (1985). /. Phys. Chem. 89, 5168-5173. Sokolov, K. v . , Hodorchenko, P. V., Bovin, N. V. and Nabiev, I. R. (1992). In: W. Kiefer, M. Cardona, G. Schaack, F. W. Schneider and H. W. Schrotter (eds), Raman Spectroscopy. John Wiley & Sons, New York, pp. 682-683. Sokolov, K. v . , Khodorchenko, P. I., Nabiev, I. R., Petukhov, A. I., Chumanov, G. I. and Cotton, T. M. (1993a). Appl. Spectrosc. 47, 515-522. Sokolov, K. v . , Byramova, N. E., Mochalova, L. V., Tuzikov, A. B., Shiyan, S. D., Bovin, N. V. and Nabiev, I. R. (1993b). Appl. Spectrosc. 47, 535-538. Spiro, T. G., (ed.) (1987). Biological Applications of Raman Spectroscopy, vols I and II. John Wiley & Sons, New York. Sureau, F., Chinsky, L., Amirend, C , Ballini, J. P., Duquesne, M., Laingle, A., Turpin, P. Y. and Vigny, P. (1990). Appl. Spectrosc. 44, 1047-1056. Taylor, G. T., Sharma, S. K. and Mohanan, K. (1990). Appl. Spectrosc. 44, 635-650. Terstappen, L. W. M. M., de Grooth, B. G., Nolten, G. M. J., ten Napel, C. H. H., van Berkel, W. and Greve, J. (1986). / . Cytometry 7, 178-183. Thomas, G. J., Jr, Prescott, B. and Olins, D. E. (1977). Science 197, 385-388. Todd, E. A. and Morris, M. D. (1993). Appl. Spectrosc. 47, 855-858.

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Todd, E. A. and Morris, M. D. (1994). Appl Spectrosc. 48, 545-548. Tripodi, D., Weis, J. W. and Bounes, P. E. (1971). Transfusion 11, 139-148. Tu, A. T. (1986). In: R. J. H. Clark and R. E. Hester (eds). Spectroscopy of Biological Systems. John Wiley & Sons, Chichester, pp. 47-112. Vakurina, T. I. (1992). Ph.D. thesis, Russian Academy of Sciences, Vladivostok. Van Duyne, R. P. (1979). In: C. B. Moore (ed.). Chemical and Biochemical Applications of Lasers. Academic Press, New York, vol. IV, pp. 101-139. Van Duyne, R. P., Haller, K. L. and Altkorn, R. I. (1986). Chem. Phys. Lett. 126, 190-196. Vo-Dinh, T., Alak, A. and Moody, R. L. (1988). Spectrochim. Acta 43B, 605-636. Vo-Dinh, T. (1989). In: T. Vo-Dinh (ed.). Chemical Analysis of Polycyclic Aromatic Compounds. John Wiley & Sons, New York, pp. 451-485. Wang, J. C. (1989). Biochim. Biophys. Acta 909, 1-9. Wiley, D. C. and Skehel, J. J. (1987). Ann. Rev. Biochem. 56, 365-372. Yu, N.-T. and Barron, B. C. (1986). In: G. Pifat-Mrzljak (ed.), Supramolecular Structure and Function. Springer-Verlag, Berlin, pp. 104-128. Yu, N.-T. and East, E. J. (1975). J. Biol. Chem. 250, 2196-2205. Yu, N.-T., Kuck, J. F. R. and Askren, C. C. (1981-82). Curr. Eye Res. A, 615-618. Yu, N.-T., Bando, M. and Kuck, J. F. R. (1985a). Invest. Ophthalmol Vis. Sci. 26, 97-101. Yu, N.-T., De Nagel, D. C , Pruett, P. L. and Kuck, J. F. R. (1985b). Proc. Natl Acad. Sci. USA 82, 7965-7968. Yu, N.-T., DeNagel, D. C , Ho, D. J.-Y. and Kuck, J. F. R. Jr (1987). In: T. G. Spiro (ed.). Biological Applications of Raman Spectroscopy: Raman Spectra and the Conformations of Biological Macromolecules. John Wiley & Sons, New York, vol. I, pp. 47-80. Yu, N.-T., Cai, M.-Z., Ho, D. J.-Y. and Kuck, J. F. R. Jr (1988). Proc. Natl. Acad. Sci. USA 85, 103-106.

10 Applications in Art, Jewelry and Forensic Science Claude Coupry and Didier Brissaud

1. INTRODUCTION The applications of Raman microscopy described in this chapter concern fields of interest as different as art, jewelry and forensic science. However, these subjects can be combined, since in all three cases the precious and unique character of the sample requires the same basic experimental conditions. The microdomain analyzed may be either a minute part of a bulk object, e.g. inclusions in gems or precise regions in a corrosion layer, which must be examined in situ, or a microscopic clue sample, such as a dust particle. Frequently, it is a microsample taken from a work of art. Depending on the nature of the object and possible constraints, the sampUng may or may not be feasible. For example, for a given painting, an art conservator may agree to the removal of a sample, imperceptible to the naked eye, at a point chosen by him, while, for an official appraisal, such sampling is often refused for the sake of the integrity of the object. Even if the operation is approved, the sample must be as small as possible. To make a comprehensive analysis of an object, it is often necessary to use several analytical microtechniques. Since Raman microspectroscopic examination requires no sample preparation and does not damage it, further analysis can be carried out on the same particle using other techniques. Therefore, only one sample is needed and it may have dimensions hardly larger than the focal spot - approximately 1 juim, depending on the aperture of the objective and the selected wavelength of the laser (see Chapter 2). This capability of obtaining, in a nondestructive way, molecular information on a microsample, makes Raman microscopy a very powerful technique. Depending on the nature of the problem to be solved and the objective of the work, the interpretation of the spectra can be made at different levels.

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Sometimes the observed vibrational Raman spectrum must be assigned in detail, but in many cases a spectral comparison is sufficient, either to ascertain the similarity of two, even unknown, compounds, or (more often) to identify the compound by reference to a database. For these apphcations, the main limitations of Raman microscopy are determined by the nature of the sample itself. Its small dimensions often dictate the use of low laser power in order to avoid thermal or photochemical decomposition. Furthermore, the frequent presence of organic compounds, such as binding media or impurities, may give rise to fluorescence interference, which sometimes completely obscures the Raman spectrum. Finally, the range of compounds which may be found, especially in art works or forensic clues, requires a large reference database. At the present time the number of pubhshed Raman spectra is relatively limited, as compared to the available X-ray diffraction or infrared data.

II. ART OBJECTS Many questions arise about art works concerning either techniques or history, as well as those involving their restoration and conservation. For some of them, the answers require the identification of the materials used, or of corrosion or decomposition products; in these cases analysis must be carried out. Consequently, analytical research is now increasingly appUed in the study of art objects. This type of work requires close collaboration between the art historian (or curator) who formulates the problem and the scientist who looks for the best way to solve it. The analytical information obtained may then surpass the requirements of the artistic world and enter the economic and social domains. In this section we should hke to emphasize that among the various analytical techniques available, the capabilities of Raman microspectrometry are particularly well adapted to the investigation of objets d'art.

A. Experimental Procedures 1. Identification In Situ As the objects of interest are unique and precious, it is often desirable to perform the Raman examination directly on them. As examples, consider the identification in situ of the yellow pigment (orpiment) in an Egyptian mummy mask (Guineau, 1989a) or the study of pigments in an illumination of a medieval manuscript (Devezeaux de Lavergne et al., 1990; Best et al., 1992). In this type of investigation the object is positioned, without

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preparation, on an adjustable microscope stage or, in the case of heavy or large objects, simply on a table. The microscope objective is then positioned as required. For a manuscript it is usually necessary to press the folio under a polymethylmethacrylate (PMMA) plate to reduce movement of the parchment due to heating by the laser beam, which may disturb the observation. Moreover, as in any optical method, it is possible to study a region through a transparent layer (Guineau, 1984). This technique is very useful, especially in the case of a layer which cannot be removed from the object, such as the glaze on earthenware, or one which sticks to it, e.g. an adhesive protective film on fragile paper. The spectrum of the opaque layer which is obtained may contain additional features arising from the transparent coating, depending on the depth resolution of the instrument. This resolution can be improved with the use of confocal illumination and observation (cf. Chapter 2). Pastels are often protected by glass, whose removal can damage these fragile drawings. Because of the thickness of the glass, it is necessary to use a long focal length objective, which has a smaller solid angle of collection (Chapter 2). The corresponding decrease in the observed spectral intensity is thus related to the thickness of the protective glass. In situ examination allows the number of analyzed points to be increased in order to control the homogeneity in a totally nondestructive way. However, this procedure increases the number of sample handlings, which is sometimes hazardous, and, in the opinion of Devezeaux de Lavergne et al. (1990), should generally be avoided. Nevertheless, this method of examination is useful for objects which can be handled without risk. Obviously, it is impossible for wall paintings, large size paintings and many precious or fragile art objects which, because of conservation constraints, must remain in a safe place, such as a museum or library. Therefore, the sampling of a microscopic part of an object is very often the best method available for its analysis. 2. Microscopic

Sampling

When it is adequate and authorized to remove a sample, two requirements have to be fulfilled: (i) the sampling must not mutilate the art work (the probe has to be as small as possible, invisible to the naked eye) and avoid locahzations where it can induce a later adulteration (such as sampHng in a dense layer which may lead to an entire collapse). The sample size needed for Raman studies, a few |xm, is compatible with this requirement. (ii) The microscopic sample must be typical and precisely related to the material to be analyzed. This criterion excludes samples of uncertain

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origin such as stains, and raises the difficult problem of restoration and repainting. The art object is first submitted to a very careful examination in order to establish suitable localizations. Its physical features are observed with the naked eye, then with a microscope. The features of interest include: color, hue and intensity, the crystalline or glassy nature of the material, the shape of crystals and heterogeneity - as well as defects such as pigments offsets or flakes, where the sampling is harmless. These observations are used for the choice of the sampling location, which help in the last identification step. The curator or the restorer in charge of the object determines whether the chosen site is safe or not. The experimental procedure employed in the collection of microsamples is determined by the class of object. Either a tungsten needle under a microscope (manuscripts, pastels and drawings) or a surgical scalpel (wall paintings and oil paintings) is used to remove the sample. The sample is usually mounted between two microscope slides, in the center of a reinforcement stuck on one shde. Flakes from wall paintings and oil paintings may be mounted in resin and cut perpendicular to the surface for a study of the different layers. The location of the sample is photographed and its position accurately recorded. The main advantage of this procedure is that it requires only one operation on the object in its storage location and in the presence of the person in charge. The sample can then be easily manipulated, studied on different spectrometers at different times, and with different excitation wavelengths in order to avoid fluorescence due to the support or the surroundings, or to confirm resonance effects. However, the principal limitation of this method is due to the possible microscopic heterogeneity and, consequently, the nonrepresentativity of the sample. Thus, the result obtained on a fragment cannot necessarily be extended to the entire work of art. With the agreement of the curator or the restorer it is possible to store the samples and develop a reference collection. Thus, new examinations can lead to the identification of impurities and minor constituents, and provide reference samples for comparison with the results of later analyses. The choice between the two procedures, in situ study or sampling, is dictated by the imperative principle of conservation and respect for art objects, which is determined by their uniqueness and their essential role in the evolution of human culture. Technological developments now in progress will soon make possible another procedure for obtaining in situ Raman spectra of art objects. The use of optical fibers to illuminate the object and collect the diffused light is combined with a compact microscope head and a spectrometer (see Chapter 3). The system is designed to operate outside a laboratory, for instance in a museum, a Hbrary or an archaeological site. This new kind of

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microspectrometer (RLFO) will effectively obviate the use of any sampling technique.

B. Application to the Study of Pigments

Pigments are the significant materials in colored layers. Consequently, their identification is of interest to art historians as a means of characterizing the artist's palette, and thus to gain a knowledge and understanding of his practice. Then, a comparison can be made between painters or workshops which can be traced through history and geographical locations. This information is particularly useful in the history of economic development, in order to estabUsh trade routes and technological evolution. Finally, the choice of a given pigment for a color, and the distribution of the pigment in a painting, may be related to social motivations. Thus, as the picture layer is valuable and its materials contain significant information, Raman microscopy represents the ideal technique for pigment analysis. 1. The Resonance Raman Effect As is generally the case for colored compounds, it is often possible to enhance the Raman scattering by pigment samples with the use of the resonance technique (see Chapter 1). When the excitation wavelength is close to that of the electronic transition of the chromophore, the resonance condition results. Some of the Raman bands may then exhibit considerable increases in intensity. In particular, certain combinations and overtones may be observed which are not detectable in the ordinary Raman spectrum. This effect is often very helpful for pigment identification. An illustration of the application of the resonance effect is the study of synthetic ultramarine (Clark et al., 1983). The color of this aluminosilicate can be blue, green or violet depending on the preparative procedure, which varies the proportions of the sulfur species, S^, S^ and, perhaps, S4, which are responsible for the color. These species absorb at 390, 600 and 520 nm, respectively. With the use of excitation at one of these wavelengths, it is possible to observe the Raman bands of the related chromophore, even when it is relatively scarce. The Raman spectrum of the silicate framework, which is present in large quantity but out of resonance, is very weak, thus allowing an easier detection of the chromophores. The enhancement of Raman overtones by resonance can also aid in pigment identification (Guineau, 1989b). For two modern organic pigments, closely related in structure, the Raman spectra are quite similar. The small wavenumber differences, which are barely noticeable on the fundamentals, are approximately doubled on the first overtones, thus allowing the two products to be distinguished.

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It is important to note that the resonance procedure requires a knowledge of the absorption spectrum of the sample. Furthermore, it may induce thermal alteration and sometimes decomposition, especially for microsamples which are inefficient thermal diffusers.

2. Reference Databank The development of a Raman spectral library for the identification of pigments in art objects involves two steps: (i) The establishment of the list of compounds used by artists, and (ii) The collection of the corresponding spectra. General or introductory works on pigments are pubUshed very frequently which provide numerous data with valuable comments (e.g. Gettens and Stout, 1966; Harley, 1982; Feller, 1986). For each art period, the technical literature is a significant source for the evaluation of painting materials and techniques. Examples are Vitruvius's and Pliny's treatises from Roman times, medieval books, such as Mappae Clavicula or Schedula Diversarum Artium or // libro delVArte de Cennino Cennini, written in 1437. Since the beginning of the eighteenth century, and especially the middle of the nineteenth century, new synthesized pigments became available which were identified by industrial patents. However, for various reasons it is often difficult to link the name of a pigment to a chemical structure: (i) The artistic vocabulary describes a color rather than a product; for example, the term 'cadmium yellow' designates cadmium sulfide or an organic substitute with a similar hue; (ii) The old recipes for obtaining a given color are often vague and sometimes employ unidentified products; (iii) The same compound may have different names according to local customs or the period. Besides colored matter, a pigment may contain additional products, impurities, or the artist's specific adducts, naturally varied, which serve to characterize it. Therefore, the list has to include a very large number of compounds. Some Raman spectra of these compounds are available from the pubUshed literature (Griffith, 1975; Guineau, 1986; Maestrati, 1989). For additional compounds, the spectra have to be recorded from reference samples. It is preferable to record the spectrum with the use of several wavelengths of excitation. The choice of reference products is not trivial, as is illustrated by the example of Naples yellow (Andersen, 1982). Three commercial samples of this pigment, assumed to be Pb2(Sb04)2 (Gettens and Stout, 1966, p. 133), were analyzed. The Raman spectrum of the first sample characterizes another lead antimony oxide, Pb2Sb206(0,OH), as confirmed by X-ray

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diffraction. The second is a mixture of the same oxide with Sb203. From the results of energy-dispersive X-ray analysis, the third sample contains, in addition to lead, not antimony, but chromium. Both PbCOs and PbCr04 have been identified in this pigment by Raman microspectroscopy. None of these samples, all of which are different, corresponds to the assumed formula. 3. Some Examples The object of the first three of the following applications is to investigate the pigments in the pictorial layer on specific supports, and to analyze the associated difficulties. In the fourth appHcation, the examination is related to the pigments in one artist's works, including those in the colored supports. All of these results are of interest to art historians and historians in general. (a) Medieval manuscripts The pigments in medieval manuscript illuminations exhibit interesting peculiarities. Most of these substances are inorganic. They are coated with a small quantity of binding material and deposited on parchment which has been previously rubbed with pumice. The samphng is sometimes aided by the presence of an offset on the opposite page. The spectra of these inorganic compounds are often intense. Furthermore, they display a very specific frequency pattern for each compound which allows an unambiguous identification to be made. However, the small size of illuminations Hmits the number of sampHngs. Therefore, in spite of these apparent advantages, few studies with this technique have as yet been pubUshed. A set of six manuscripts which were illuminated during the twelfth century in an abbey in the north of France and conserved at Paris (Bibhotheque Nationale) were examined (Guineau et al., 1986). The blue color was studied in different kinds of initial, historiated, decorated or simply colored, which showed variable hues, pure blue, gray, and sometimes violaceous, with different levels of saturation. About 40 microsamples were collected in the hbrary in the presence of the conservators. Raman spectra (Fig. 1) obtained from all of the samples show common features: they are dominated by a strong band at 548cm~\ with weaker bands at 260, 585 and 1098 cm~^, sometimes with various additional bands. This group of four bands is characteristic of ultramarine blue, an aluminosiUcate, and reveals the presence of only this pigment. For pale samples, there are no other bands which would indicate the addition of white pigments, e.g. chalk, white lead or gypsum. In the blue-gray samples, microscopic observation shows tiny black particles which are identified by their Raman spectra as graphite.

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O

3 u < > <

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WAVENUMBER (cm"^) Figure 1 (a) Raman spectrum of a sample from a twelfth-century manuscript (Guineau, 1986). (b) Reference spectrum of ultramarine blue from lapis lazuli.

The interpretation of these data leads to very interesting conclusions. The ultramarine is extracted from the semiprecious stone, lapis lazuli, which, in the Middle Ages, came from mines in Badakashan, a province in what is now Afghanistan. Its very early utilization in Western manuscripts is revealed by this result and confirms its trade along routes which have yet to be studied. The painting practice of the abbey workshop is specified: the use of only one pigment, whatever the situation (and whoever the painter or the scribe), which was more-or-less diluted in the medium in order to change the hue, or blended with graphite to obtain dark blue shades. For most of the samples the spectra display only the Raman bands corresponding to S^ (260, 548 and 1098 cm~^), the most abundant species

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WAVENUMBER (cm"^) Figure 2 Raman spectrum of a pale-yellow particle in one sample of a twelfth-century manuscript (see text). The bands marked p are due to ultramarine blue.

responsible for the blue color, and S^ (585 cm~^). The lack of evidence of impurities indicates careful purification of this expensive material. In one sample, a pale-yellow particle was observed among the blue ones. The spectrum obtained with the laser beam focused on this spot (Fig. 2) has two additional bands, one at 465 cm~^ (strong) and the other 208 cm~^ (weak) which allow the particle to be identified as quartz. All of the samples of the same manuscript exhibit the band at 465 cm~^, indicating that they all contain very tiny specks of sand, although they are not seen under the microscope. The ultramarine used for the decoration of this codex has a lower level of purity. The spectra of some samples, from different manuscripts, rather violaceous in hue, present extra bands at 350 and 685 cm~^ which are assigned to the chromophore S4. The presence of this species, which gives these samples their special hue, characterizes a different pigment quality. The contribution of the Raman identification of pigments is not limited to technical knowledge. Under St Bernard's influence, the decoration of manuscripts at Citeaux (France) changed into a monochromatic, austere

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700

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WAVENUMBER (cm"') Figures Raman spectrum of a sample from a fresco at Saint Germain (Auxerre, France). style, although the blue pigment used was not modified by the new tendency and remained, unexpectedly, the same costly ultramarine. Ultramarine was identified (Coupry, 1991) in a manuscript written c. lOOOAD in the abbey of Saint-Germain (Auxerre, France). On the dedication image only the patron saint of the abbey has a garment covered with this rare and expensive pigment, thus emphasizing his importance. A wide range of pigments was analyzed in situ in a historiated initial from a sixteenth-century German manuscript (Best et al., 1992). In a dark gray mixture, it is possible to identify three main products, as well as small amounts of three others. This result illustrates the capability of the technique to deal with mixtures, which are generally difficult to study and were commonly used in periods when the range of available pigments was limited. (b) Wall paintings Wall decoration requires products for the preparation of the wall, as well as for the painting, according to the chosen procedure a fresco or a tempera (Mora et al., 1977). The study of either category of product may yield interesting information. Both types have been investigated at Saint-Germain (Auxerre, France). The crypt of this abbey has the oldest Carlovingian frescoes in France, which date from the mid-ninth century. They include historical scenes, inscriptions and simple decorations of strips and leaves. The architecture of the church has been modified several times, with the addition

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of new spans and a gothic choir, which involved changes in the crypt and new paintings. The analysis of pigments and techniques is one means used by archaeologists, along with excavations, examination of historiated texts, and studies of elevation and rising, to understand the transformation of the construction. In a study by Ferrer (1991) samples were taken from different decorations. Some were in well-dated locations, the ninth century or Gothic period, others were in areas of unknown dates. They were always red in color. For most of these samples, only the pigment was examined. A mineral pigment was invariably present, mainly haematite in the Carlovingian period, or vermilion (HgS), at later times. The very intense spectrum of HgS was easily obtained with the use of an He-Ne laser (A = 632.8 nm). However, the iron oxide presented some difficulties, as it is a poor Raman scatterer. The latter sample may be a mixture of different iron oxides. The spectrum (Fig. 3) shows the presence of hematite, Fe203, with bands at 220, 295 and 410 cm~^. The intense bands at 613 and 652 cm~^ suggest the presence of FeO (or Fe304) (Thibeau et al., 1978). Neither lead compounds nor organic dyes have been found in the Carlovingian samples. These classes of compound have rarely been analyzed by Raman spectroscopy, although a very strong fluorescence is often an indication of their presence. The fluorescence observed with these samples is not uniform and is probably an indication of the presence of organic dusts or microorganisms which have grown on the exposed walls. Samples are often mounted in resin in order to study the different layers. Figure 4 shows the structure of a sample from Auxerre. Under a strip colored by Fe203 there is a thick white layer with yellowish transparent grains. They are sand grains, perhaps coming from the mortar, but it is possible that they were added to the whitewash itself, as they are identified in several samples. The whitewash layer is a mixture of at least three compounds. The spectral analyses of different zones show that they are present in variable proportions. The whitewash is customarily prepared from a lime and gypsum base. Organic dyes are sometimes used for wall paintings. Their identification in Roman paintings will be described below, in the section concerning the Raman characterization of dyes.

(c) Easel paintings The capability of Raman spectroscopy to distinguish different crystallographic structures of a compound has been exploited in the examination of contemporary paintings (Coupry et al., 1987) in order to propose an authentication test. Titanium white (Ti02) exists in two crystaUine forms, anatase and rutile. The development of available products can be followed by reference to industrial patents. Thus, the pigment can be used as a chronological indicator.

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WAVENUMBER (cm~^) Figure 5 Raman spectra of Ti02. (a) Anatase. (b) Rutile. The Raman spectrum of each product (Fig. 5) exhibits strong bands at very characteristic wavenumbers, which leads to easy identification. However, the binding media, which are especially complex in easel paintings, may be strongly fluorescent. In a study of paintings by contemporary artists at the Musee d'Art Moderne (Paris), X-ray microfluorescence was employed by the Laboratoire de Recherche des Musees de France (Paris) to detect the areas which contained titanium. Then, selected samples from these regions were analyzed with the use of Raman microscopy. Fortunately, the medium used by Picasso, Pollock, Dubuffet, etc. in the paintings which were analyzed, yields only a weak fluorescence and its Raman spectrum is weak or shifted in frequency from that of the pigment, which was characterized without difficulty. An interesting investigation could be made on these paintings by pinpoint analysis of cross-sections. (d) Synthetic pigments Since the nineteenth century more and more synthetic pigments have appeared. A study is in progress of paintings by Constantin Guys, a French

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WAVENUMBER (cm ') Figure 6 Raman spectrum of Prussian blue from a drawing by Constantin Guys (see text).

artist of this period (Dufilho and Guineau, 1988; Dufilho and Coupry, 1992). His palette is limited - centered around blue, with various hues. The Raman spectra of a large number of blue samples corresponding to various hues allow three compounds to be identified: Prussian blue, cobalt blue and ultramarine blue. The first, ferric ferrocyanide, Fe4[Fe(CN)6]3, is the earliest of modern pigments (beginning of the eighteenth century). The most intense band in its Raman spectrum, at 2150 cm^ (Fig. 6), is assigned to VQ^. Some samples yield additional bands of lower intensity in the same region, which are probably due to vibrational frequencies of the CN ligands in similar compounds (Bertran et aL, 1990). Cobalt blue is cobalt aluminate, which was discovered in 1802. The spectrum (Fig. 7), which is the same for all samples, is comparable to that of the present commercial product, indicating a great constancy in the preparation of the pigment. The spectrum of ultramarine blue is the same for the natural or the artificial compound (cf. Fig. 1). It always displays the bands of S^ and S^, but, depending on the sample, the bands due to the species S4 can be strong (Fig. 8), or totally absent. Differences in the spectra probably reflect variations in the manufacturing process. A comparable variety of coloring compounds can be found for the support material. For example, most letters written by C. Guys were on blue paper from which fibers can be picked out. Microscopically, some are uniformly colored; their weak Raman spectra are characteristic of Prussian blue, which was used as a dye. Other fibers show blue, rounded, sub-|jLm particles, which are identified by Raman spectroscopy as ultramarine blue. From the size and

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WAVENUMBER (cm"^) Figure 8 Raman spectrum of ultramarine blue. the form of particles, it is concluded that this product is the artificial ultramarine blue discovered by Guimet in 1831. The industrial production of this compound began in 1830. Thus, these results are useful not only to study the practice of the artist, but also to establish the history of the pigments. The new synthetic pigments have been increasingly employed by both artists and industry.

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WAVENUMBER (cm"^) Figure 9 Raman spectra of indigo, (a) From a drawing by Mignard (see text); (b) reference spectrum.

C. Dye Studies

Although some dyes are good Raman scatterers (Best etal., 1992, refs 6-12), their Raman spectroscopic identification in art objects is often difficult for several reasons. They are fragile organic compounds; therefore, the power of the laser excitation must be low enough to avoid decomposition or other modification. The dye layer is often thin; consequently, the amount of product is limited and the spectral intensity is weak. Natural dyes, used until the nineteenth century, are mixtures of several compounds, which complicates the analysis.

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An important red dyestuff is madder, composed chiefly of alizarin and purpurin, di- and tri-hydroxyanthraquinone, respectively. It can be used as a coloring matter for textiles or be prepared on a base, like a lake. It can be identified in either state. The Raman spectra obtained directly on a wool fiber from the binding of a medieval manuscript (Guineau and Guichard, 1987) and on a sample of a Roman wall painting (Guichard and Guineau, 1990) show only three bands. Their pattern is close to that in the spectrum of alizarin. In spite of the intensity increase of some modes which can be achieved with the resonance Raman effect, the observation of these bands is hindered by a very strong fluorescence. The presence of alizarin was confirmed by a SERS study (see Chapter 8) after extraction of the dye from the support, wool or alumina for wall paintings. When a dye is concentrated enough, and when fluorescence does not perturb the Raman spectrum, it can be identified unambiguously, e.g. the study of indigo on paper fiber (cf. the following section). In a pastel drawing by James Ensor (Guineau, 1989b), the presence of a synthetic dye, methyl violet, a triarylmethane compound, was detected in the form of minute inclusions (<10 |xm) in a chalk particle.

D. Supports for Restoration and Conservation To determine the procedure to be used for a restoration it is necessary to determine the nature of the initial or altered products in the damaged zone. Then, the cause and the mechanism of the damage can be established. As an example, a drawing by Bronzino, on blued paper, showed large white spots. The blue color in the paper results from a preparation deposited on the back of the paper. A microscopic sample chosen from an unadulterated area exhibits tiny dark particles in a white material. The Raman spectrum of each part indicates graphite and lead white (PbC03), respectively, with no evidence of a blue compound (Coupry and Revault, 1991). For the restorer, this incomplete identification is sufficient, as it is concluded that the lead white is responsible for the damage. Another example is the analysis of the deterioration of stonework by lichens. These microorganisms build up encrustations as a result of the chemical action of the oxahc acid which they secrete. To identify the nature of encrustations in Renaissance frescoes (Palazzo Farnese, Rome), samples of painted and ground layers in damaged areas, as well as the ground layer in undamaged ones, were analyzed (Edwards et al., 1991, 1992). The Raman spectroscopic results show that encrustations contain mainly calcium oxalate monohydrate, with, for some samples, included particles of the ground layer and/or unknown, possibly organic, compounds. Once the cause of alteration is known, the restorer must be sure that the chosen process does not introduce difficulties in other areas. For example, in

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Figure 10 (a) Spectrum of a forsterite inclusion in diamond; (b) reference spectrum of forsterite (AQ = 514.5 nm). a drawing by Mignard on a blue support (Letessier, 1991) white fibers had become yellowish. Before attempting to bleach these fibers, it was essential to know whether the process was safe for the dark blue fibers mixed in the paper. In this case the answer was positive, as the Raman spectrum (Fig. 9) obtained in situ on a fiber, identified the dye as indigo, a very stable dye which is able to endure the bleaching process. E. Conclusion These examples of applications have shown that Raman microspectroscopy is a very useful tool for the investigation of art objects. Although it was first used for identifying pigments, its range of application has broadened to include dyes, corrosion products, etc. Significant advances in Raman instrumentation, as well as the increasing number of pubhcations available on reference compounds, will allow more specific investigations of art objects to be made. Nondestructive analyses by Raman microscopy now provide information on materials, practices and alterations, information which is essential for art historians, conservators and restorers. III. JEWELRY Certainly a very attractive application of Raman microscopy is the study of jewelry. This method of characterizing gems by the identification of inclusions

Art, Jewelry and Forensic Science 439 was suggested early in the development of micro-Raman instrumentation (Dhamelincourt and Schubnel, 1977) and was demonstrated the following year (Dele et al., 1978). The chemical nature of inclusions varies according to the genesis or synthesis of the gems. It indicates clearly their natural or artificial origin and, in the latter case, may suggest processing methods. For precious stones this technique may provide an indication of their geological, or even geographical, origin.

A. Experimental Procedures 1. Samples The identification of inclusions is carried out in situ, without any preparation of the gem, whether it is rough or shaped. This investigation is independent of the size of the inclusion, from one to several hundred ixm^, as well as that of its physical state, solid or fluid. The inclusions may be investigated at depths of up to 3-5 mm from the surface with the use of low numerical aperture microscope objectives. Thus, the poHshing operation, which is necessary for other analysis techniques in order to bring the impurity close to the surface, can be avoided. The micro-Raman analysis can be performed through a crystalline face or a cut facet. 2. Spectra The laser beam is focused directly through the host into the inclusion. The only condition on the experiment is the transparency of the host. The observed spectra exhibit bands of the inclusion, as well as those of the gem. The latter are easy to identify by reference to the spectrum of the gem alone, although they may mask the spectrum of the inclusion, and thus hinder its characterization. The Raman spectra of gems exhibit various interferences. For example, diamond presents only a sharp, intense band at 1332 cm~^. Rubies and sapphires, corundums colored by chromium and titanium, respectively, have the spectrum characteristic of AI2O3, with bands at 418 and 644 cm~^. Emerald, a beryl Be3Al2Si60i8, has a crowded spectrum, with two main bands at 687 and 1070 cm~^. Thus, the observation of the spectrum of a hydrocarbon inclusion in emerald below 1100 cm~^ becomes difficult (Dele-Dubois et al., 1981a). Similarly, the spectrum of a graphite inclusion in diamond can be masked by overlapping of the single band of diamond at 1360 cm"^ (Dele-Dubois et al., 1981b). In this type of investigation the wavelength of the excitation must be carefully chosen to reduce the fluorescence of the inclusion and, particularly, that of the host lattice. For example, the intrinsic fluorescence of rubies.

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natural or synthetic, over the range of 640 to 745 nm is well known (Dubois-Fournier, 1989). This fluorescence is due to the presence of chromium ions. The observation of the spectra of inclusions in rubies is very difficult with the use of red excitation, especially if the inclusions are deep within the host. In general, it is necessary to choose the wavelength of the excitation to minimize its absorption, as well as that of the Raman scattering by the host, which is often a colored compound. When the inclusion is composed of a mixture of several compounds, the identification of each may be difficult. Here again, a large databank of reference Raman spectra is essential. The spectra of many minerals have been assembled in a recent review (Schubnel et al., 1992).

B. Analysis of Inclusions 1. Natural Minerals Inclusions of various types may be included in gems - sometimes even several different ones in a given gem. The origin of these inclusions is linked to the formation or growth of the stone. The inclusions may be altered by such factors as structural instability and temperature. Exolution of inclusions can also occur, especially in corundum. In certain cases inclusions can be very complex. For example, an inclusion of apatite has been identified inside an inclusion of zircon, which is in turn inside a Colombian sapphire. The Raman spectra exhibit the presence of one, two or three compounds, depending on the point of analysis. Furthermore, spectra obtained within an inclusion often indicate variations in composition. Thus, as examples, the shift of the band characteristic of chromite at approximately 690 cm~^ is a measure of the concentration of chromium. An inclusion in hematite was found to contain at various points other iron oxides, FeO or Fe304. Fluid inclusions are not very common. However, in a given diamond numerous blackish particles were identified as graphite by the band at 1580 cm"^ (the other one was masked by the spectrum of the gem). In addition, a fluid inclusion was identified as that of gaseous nitrogen by its characteristic band at 2342 cm" ^ The Raman spectrum of another fluid inclusion was observed in an emerald from Muzo (Columbia). It is a biphase composed of gaseous N2 and CO2 and a Hquid phase of saturated hydrocarbons (Dele-Dubois et aL, 1981b). Most inclusions are solid; thus, silicates are identified in diamonds from South Africa, as well as inclusions of forsterite, Mg2Si04 (Fig. 10), diopside CaMgSi206, pyrope, Mg3Al2Si04, calcite (Dele-Dubois, 1986a), apatite, etc. The nature of inclusions in gems depends on their geological origin (Dele-Dubois et al., 1993a). Rubies extracted from volcanic lodes, such as

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those from Thailand or Cambodia, contain pyroxene, while Burmese rubies can be identified by inclusions of calcite, amphiboles and metamorphic carbonates. Metasomatic steams produce inclusions of quartz and apatite, as found in Vietnamese rubies. Regular-shaped, opaque inclusions have been observed in several Burmese rubies (Dubois-Fournier, 1989). The spectra obtained (Fig. 11) are similar to those of the iron sulfides, pyrite and pyrrhotite. This result suggests the presence of a nonstoichiometric compound, with a composition between those of FeS and FeS2. One inclusion yields the superposition of the same spectrum with that of sulfur (bands at 151, 220 and 471 cm~^), in support of the hypothesis of the existence of an iron sulfide which is partially degraded, with the formation of sulfur. Zircon, ZrSi04, is associated with many minerals. However, it is not a geological tracer, although its presence validates the natural origin of the gem. The inclusions which have been identified by Raman spectroscopy are reported according to the nature of the gem, e.g. diamonds, emeralds and rubies, sapphires (Dele-Dubois et al., 1981a,b, 1993a; Dele-Dubois and Schubnel, 1987; Schubnel etal, 1992).

2. Synthetic Gems The nature of inclusions in synthetic gems is determined by the method of production; thus they are very different from those found in natural minerals. The identification of inclusions in synthetic gems provides a method of determining their origins and, in some cases, their authenticity (Dele-Dubois et al., 1986a). Here again, Raman microspectroscopy is a well-adapted technique (Dele-Dubois et al., 1993b). Emeralds and rubies are synthesized by a flux-fusion method or a hydro thermal process. In the case of emeralds the two methods lead to the formation of inclusions of phenakite, BeSi04, which is easily identified by the Raman bands at 876 (^-Si-O), 914^920 and 948 cm~^ (Fig. 12). Phenakite is associated with water in the hydrothermal process. On the other hand the flux-fusion method leads to solvent inclusions, namely polymolybdates in Chatam, Gilson and Igmerald emeralds. In the Lennix emerald, which is synthesized by the flux-fusion method, the inclusions are identified as beryl and quartz, and sometimes as orthorhombic or hexagonal molybdenum oxides, M0O3 (Fig. 13). The metallic aspect of these oxides previously suggested the presence of platinum (Dele-Dubois et al., 1986b). Similarly, rubies which have been prepared by flux fusion contain traces of solvent such as polymolybdates and orthovanadates. The spectra obtained from different inclusion spots may exhibit differences due to inhomogeneous solvent mixing or selective trapping during crystallization (Dubois-Fournier, 1989).

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IV. FORENSIC SCIENCE A. Introduction There is an increasing tendency in jurisprudence to replace testimonies or circumstantial evidence by material clues. Thus, if a person commits a crime, there are invariably materials which he leaves behind as evidence of the act. The concept of evidence is the basis of criminology, which may be defined as the art and science of discovering, analyzing and identifying clues. The

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WAVENUMBER (cm ') Figure 12 (a) Spectrum of a phenakite inclusion in emerald (E indicates Raman bands of emerald); (b) reference spectrum of phenakite (AQ = 488 nm).

evidence must be obvious, convincing and definitive. It is, then, through the identification of material traces - clues - that justice will proceed to reveal the perpetrator of the act.

B. Methodology

For juridic reasons any clue constitutes an element which may be able to provide conclusive evidence. Therefore, it cannot be destroyed, or even damaged, by the analyses employed. Forensic science can then use Raman spectroscopy as an analytical technique, at even microdimensions, which preserves the integrity of the sample (Andersen, 1982). Many different types of sample may be involved, depending on the origin, e.g., (i) biological: blood, semen, urine skin hair (natural or textiles), feathers vegetal residues, e.g. fibers, wood, paper

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The methodology used for either compound identification, or simply the proof of similarity or differences between different compounds, is based on a rigorous comparison of the experimental results obtained from clue samples with analogous reference data. It is thus important in the present application to establish a catalog of reference Raman spectra. An example is sufficient to illustrate the necessity of the reference data bank. In a rape case, a reference to the databank allowed the nature ^nd chemical composition of small ungual samples to be determined, without any previous knowledge of their constitution. The databank which was compiled was the sum of a number of smaller spectral collections, with a common format. It should be emphasized that such a databank is an indispensable tool for the forensic scientist, as it must, as indicated, be apphcable to a wide range of products. They are, in fact, everyday elements, rather than laboratory chemical compounds. Once the Raman spectra of the clues have been obtained, the problem which follows is the choice of the bands used to interrogate the databank. The sample investigated may be a simple constituent or it may be composed of a number of different substances. The proofs that the forensic scientist can present in a legal process are either that two clues can be shown to be identical, or that it can be demonstrated that they are different. These facts can often be determined without identification of the sample or the various chemical compounds which may be included.

C. Instrumentation

The Raman microspectrometer used in this work has been developed to respond to the specific needs of forensic science. It is a modified version of a standard instrument, the DILOR XY, which is currently employed in macroand micro-Raman spectrometry, as well as in fluorimetry. The use of a microscope, which focuses a laser beam on the sample, not only allows the analysis of small samples to be made, but also can serve to perform pinpoint mapping of the investigated area. The nondestructive analysis of the sample can be assured by a proper choice of the wavelength and intensity of the laser excitation. Not only are the samples of various origins, they also are of various sizes and forms. For example, clues can be obtained from such varied sources as car bumpers, passports, works of art, etc. The spatial positioning of the sample under analysis may often be important. Thus, the microscope stage is modified so that its three-dimensional displacement allows the examination of any point in the sample to be made.

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D. Some Examples of the Use of Raman Spectra as Juridic Evidence Basic to criminology is the principle that, 'a person cannot disappear without leaving any trace'. Thus the elements of interest in criminal investigation are usually found in the area of the crime and are microscopic in nature (Bisbing, 1989). Raman microscopy can often be exploited in the identification of these elements, either alone, or with the aid of other analytical techniques. 1. Paintings One of the most important applications of Raman spectroscopy in criminology is the identification of organic and mineral pigments used in various paints. With the aid of resonance and a suitable choice of the excitation wavelength, the Raman spectra of the pigments of interest are enhanced with respect to the spectra of other components, binder, solvent, etc. Often the Raman resonance effect permits the identification of one or several chromophores present in only a few per cent in a paint sample. The techniques which can be employed with a Raman microprobe are especially important in the analysis of objets d'art, where the pigmented layer can be directly analyzed, independent of the nature of the support (see the previous sections of this chapter). Pigments can be divided into natural and synthetic colors; their histories and techniques of fabrication are usually known. A comparison of the presumed date of a given work of art and that of a pigment identified by spectral analysis can provide evidence of an anachronism, thus reveahng a falsification. Raman microspectroscopy appears to be the most valuable analytical method among those which are accepted in the forensic sciences. As an example, consider the analysis of some pastels. Normally the analysis begins by an investigation of the works' history, e.g. presumed date of realization, artist's techniques, supports and pigments employed at that time, and so on. In this case the pastel is presumed to date from 1909. The next step is usually an in situ examination of the painting. In the present case the work was placed directly on the microscopic stage and the spectra of the blue and white pigments, which are usually dominant, were then recorded (see Fig. 14). As Raman microscopy is essentially a punctual method, it is necessary to take spectra from a number of different points. This procedure yields information concerning the spatial distribution of the identified pigments. In this example the blue color was determined to be ultramarine blue, a mineral pigment, either pure or mixed with phthalocyanine (PB15), an organic pigment. Ultramarine blue, which was originally a natural pigment, was later synthesized. It cannot, therefore, serve as a date indicator, as it has existed since Antiquity. On the other hand, free phthalocyanine was synthesized for the first time in 1928. This blue organic pigment (PB15), as well as its green, copper derivatives (PG7 and PG36), have been marketed since 1935.

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Clearly, the presence of phthalocyanine or its derivatives limits the date of the painting. In this example, the work could not have been created in 1909 with pigments which became available in 1928! In some cases,fluorescenceeffects are observed. They are usually caused by glazes over the surface of the picture. It is then necessary to make initial observations with an ultraviolet lamp in order to localize areas which are relatively free of fluorescence. Unfortunately, because of fluorescence interference, the choice of pigments which are useful for analytical purposes is quite Hmited. However, the use of excitation in the near-IR region can usually eliminate the fluorescence problem (see Chapter 3). Depending on the pigment and the nature of the glaze it is sometimes possible to identify the pigment, even under a fluorescent glaze layer, after a long exposure to laser light. This method is effective if the visible absorption is particularly intense, as in the case of Ti02, the most common white pigment. 2. Paints When paint samples have been taken at a particular site, they do not necessarily have to be preserved. These samples can then be examined by X-ray fluorimetry or infrared spectroscopy. The first of these techniques, however, does not detect pigments of organic origin, while the latter is more sensitive to the binding medium than to the chromophore, considering the relative proportions of these two products. Raman spectroscopy is thus a technique which is more important in the detection and identification of pigments of organic origin, in particular with the aid of resonance enhancement. The pigment composition of a paint trace can be determined and compared with the composition of a reference paint. For paints with the same apparent color, they can be distinguished by the presence of different pigment shades, often in very weak concentrations. As an example, consider the repainting in green of a stolen car. The microscopic observation of the clothing of a suspect revealed micro-splashes of green paint. Raman spectroscopic comparison of the paint samples found in the suspect's shop, as weU as the paint on the stolen car, with those found on his clothing, provided conclusive evidence. Furthermore, with the aid of a spectral databank the specific composition of the green paint was determined. It was composed of an organic blue pigment (phthalocyanine PB15) and a mineral yellow (lead chromate). 3. Polymers Like paints, polymers represent a large domain where Raman spectroscopy can provide information in the field of poUce investigation. At the present

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time polymers are widely used in industry. It has thus been necessary to establish a reference library of the vibrational spectra of the most commonly used polymers, e.g. polyurethane, polyethylene, polyvinyl chloride and polyesters (see Fig. 15). The role of Raman spectroscopy in this area is principally to confirm or to improve the results obtained by other techniques, such as infrared spectroscopy or melting point analysis. 4. Minerals Physical chemical methods such as X-ray diffraction or fluorescence are often insufficient to estabhsh the nature of a mineral. The limitations are usually due to problems of sample preparation, rather than to the analytical method itself. Raman microscopy can offer a complement to these other techniques, particularly as it can be used to analyze selectively a microcrystal in a mixture.

E. Quality Control

This type of investigation can aid in the design and fabrication of a product. Thus its weak points can be identified and sometimes rectified. Furthermore, as it is then well known to the juridic services, possible falsifications or counterfeits can be more easily identified. Administrative and other inviolable documents are increasingly fabricated with the use of polymeric materials. Thus, Raman microscopy provides an in-depth method of analyzing official papers such as identity cards, residence permits, passports, etc. In some cases various sheets in laminated structures can be investigated.

F. N e w Areas of Application

In the forensic sciences there are often problems which are difficult to resolve by classical analytical methods. Other techniques must then be developed. The following two examples are among those in which Raman microscopy will probably become very important. 1. Drugs Although there exist a number of chemical and physicochemical methods for the identification of drugs, Raman microscopy could be essential in the analysis of dust taken from personal objects of a suspect (Hodges and

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WAVENUMBER (cm'') Figure 15 Raman spectrum of a polyester (AQ = 514.5 nm; laser power = 10 mW). Akhavan, 1990). Such minute samples may contain drug particles which can be identified by this technique (Fig. 16). 2. Propellants and Explosives Small quantities of propellants and explosives have been detected and identified with the use of Raman microscopy (Jenner et al., 1982; Carver and Sinclair, 1983; Hodges and Akhavan, 1990; Akhavan, 1991). Even picogram quantities of these materials have been analyzed (see Fig. 17). Most of these investigations have been focused on these products or their constituents before combustion or explosion. Unfortunately, in the forensic sciences the decomposition products of these substances are often more significant. Such residues can be detected on the hands or clothing of a suspect. These organic compounds, which are primarily the energetic constituents of the original product, are at least partially burned. Some preliminary studies by Raman microscopy of unburned solid residues resulting from these processes are suggestive of a method of reliable investigation in this domain. G. Conclusions

Micro-Raman spectroscopy is a method which provides an effective answer to the needs of the forensic sciences. The principal points to be emphasized in this application are: (i) The enormous variation in the nature of the samples to be investigated, (ii) The preservation of elements under seal, which represent material evidence of a criminal act, and (iii) The analysis of microquantities of substances, clues that must not be destroyed.

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Figure 16 Raman spectrum of cocaine chlorohydrate (AQ = 514.5 nm; laser power = 0.1 mW).

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WAVENUMBER (cm-^) Figure 17 Raman spectrum of nitroglycerine (1% in KBr; Ao = 514.5 nm; laser power = 500 mW). Considering the variety of Raman microscopic applications, there are still many areas to be explored. However, in the present context it should be noted that precise details concerning the experimental procedures, as well as certain bibliographical data which are employed in the forensic sciences, rest in the domain of classified information.

REFERENCES

Akhavan, J. (1991). Spectrochim. Acta 47A, 1247. Andersen, M. E. (1982). Microbearn Analysis, 197. Bertran, J. F., Reguera-Ruiz, E. and Pascual, J. B. (1990). Spectrochim. Acta 46A, 1679.

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Index

(ff; folios following) Abbe invariant rule 37 sine condition 82 Aberration spherical (see spherical aberration) window 67ff Absorbing sample 34, 245 Acceptance angle 38, 44, 128 cone 128 Achromat 214ff ADC 161ff Adsorbed species 234ff Aerosols 301 marine 299ff Aging 371 Airy disc 53 function 96 Al2Si05 293, 305 Albite 298 Aliasing 160 Alizarin 437 Alteration (of artworks) 437 Aluminosilicates 343ff Amorphization 260ff pressure-induced 315ff Amphiboles 295, 441 Analog signals, sampUng of (see sampUng) Analog-to-digital conversion (see ADC) Analysis, elemental and molecular 223ff, 369ff Analyzer 13ff, 270, 282 Anatase 294 Andalusite 293

Angle (see also acceptance angle) of collection 29, 39 of incidence 28 Anhydrite 300 Anisotropic (see bandshapes, anisotropic) Annealing 260ff Anorthite 298 Antarticite 323ff cokes 313ff Anti-Stokes spectrum 3ff, 7, 17 Apatite 322 Aperture 38 matching of 106ff numerical 28, 44, 58, 129ff, 207ff Aplanetic points 68ff, 376 Apodization 167ff Aqueous phase 321ff Aragonite 295, 306 Array intensified 118 photodiode 116 Artworks 442 alteration of 437 authentication of 431, 441, 446 Aspherical mirror (see mirror, aspherical) Astigmatism 91ff Authentication (see artworks)

Background 220 Backscattered electrons 204ff Backscattering configuration 29 geometry 12, 14, 238 Band shift 259, 265, 335 Band width (see FWHM) Bandpass, spectral 82ff

456 Index Bandshapes anisotropic, in amorphous solids 15, 19 in amorphous soUds 19ff isotropic 15 Beam quaUty, Laser 56ff Beam waist 54ff BeamspHtter 32, 211ff, 227 dichroic 105 Beer's law 6 Bicarbonates 321ff Binning 112 Binomial smoothing {see data smoothing) Bioaccumulations 370ff Biochemistry 367ff BiodegradabiHty 371 Biology, biological 202, 223ff, 367, 375 Birefringence 330ff Bisulfate 321 Blue compounds {see cobalt blue, indigo, phthalocyanine, Prussian blue, ultramarine) Boron nitride 250 Boson band 313 Bremstrahlung 204ff, 220 Brightness 37, 43 Brillouin scattering 218 Bronsted acidity 247 Brookite 294 C - O - H - N - S system 329 Ca(OH)2 316 Calcination 246 Calcite 295, 300, 306 Camera lenses 77ff Cancer cells 379 Carbon 320 materials 256 soot 300 Carbon dioxide 319ff, 330ff Carbon monoxide 332 Carbonates 306, 321ff, 372 Carbonyl group 10, 282 Carotene, j3 396 Carriers 273 Cassegrain objective {see objective, Cassegrain), electron probe {see electron probe) Cassiterite 339 Catabolism 370ff Catalysts 244 Cataract 391 Cathode luminescence 205, 214ff

CaTiOs 305 Caustic surface 63, 91 CCD 111, 184, 266 detectors 175, 290ff Cell diamond anvil 259ff, 316 ephitheUal 373 high-pressure 261, 304ff K562 405 living 206, 367, 379, 389ff, 409ff physiology of 367, 374 ultrastructure of 367ff Cellular pathways 367 scale 371 Ceramics 250 CH4 {see methane) Charge-coupled device {see CCD) Charge-injection device {see CID) Charge-transfer devices {see CTD) Charge-transfer efficiency 113 Chemical composition, analysis of 280 Chemisorption {see also adsorbed species) 247 Chitin 373 Chondrites 296 Chromatography high-performance 233ff liquid 231 micro- 23 Iff thin-layer (TLC) 231 Chromites 295, 440 Chromophore 425 Chromosome 389ff chromatin 368 CID 115 CO {see carbon monoxide) CO2 {see carbon dioxide) Coatings protective 255 silica 257 Cobalt blue 434 Coesite 299 Collection efficiency 44, 58ff, 134 Colored samples 23 Iff Coma 91 Compensating lenses 67ff Condensed gases {see gases, condensed) Condenser 202ff Conduction materials, inorganic 278 Confocal configuration 40, 48, 145, 208, 374

Index 457 diaphragm 40ff effect 42, 47 line scanning 185 microscope {see microscope) pinholes 40ff Conformation 282 Conservation (of artworks) 437 Contrast 208ff Convolution 154ff Copolymer 280 CORALIS {see line scanning, confocal) Coverglass 66ff Cristobahte 296, 316 Critical angle 128 Crocetine, dimethyl 396 Cross-over 202ff, 225 CrystaUinity 262, 284 Crystallographic orientation 267 CTD 110 Cut-off 101 Cuticle 373 Cytoplasm 371, 374

Differential scattering cross-section, Raman 329, 332ff Diffraction limit 28, 368 Diflubenzuron 373 Digestion 371 Digital image {see image, digital) Diopside 440 Dipole moment 7 Dirac function {see impulse function, sampling function) Direct imaging {see imaging, direct) Dispersive Raman spectroscopy 125ff Divergence, asymptotic 130ff DNA 383ff Dolomite 307, 340 Dosage level 261ff Doxorubicin 395 Drawings 437ff Drugs 449 Dry objectives {see objectives, dry) Drying 245 Dyes, synthetic 436, 437

Dark current 114 Data acquisition 150 digital 165 sampling 150 smoothing 152ff, 169ff Databank 426, 440, 445 DDT 373 Defects 279 Deflection, laser 183 Dehydration 246 Delta function {see impulse function) Density of states 307ff Depolarization ratio 16, 31ff, 276 Depth of focus 33, 36, 42, 54, 146ff Depth profile 187, 260 Detector 37 germanium 108ff, 119 InGaAs 108, 119 multichannel 41, 108 photoelectric 106ff single-channel 107 sohd-state 108ff Detoxification 371, 375 Diamond 299, 319, 439ff diamond-anvil cell {see cell, diamond-anvil) Diamond-like carbon (DLC) 255 Diaplectic glass 296

Earth sciences 289ff Easel paintings 431 Ecology 371, 375 Edge filter {see filter, edge) EDS 204, 221ff EELS 205, 370ff Electric field 6ff, 16, 52 internal {see internal field correction) Electric vector 28 Electromagnetic radiation 5ff Electron beam 202ff microscope, microscopy {see microscope, electron) probe 203, 214ff, 369ff EUipticine 396, 405ff Emerald 439ff Encoding 188ff Enhancement: chemical 382 electromagnetic 382 Environmental sciences 289ff Epitaxy 254, 276 EPMA 204, 221ff, 371ff Etendue 81, 89, lOOff geometric {see optical extent) Evolution 372ff Excitation focusing of 27

458 Index Excitation (cont.) polarization of 28 profile 23 pulsed NIR 121ff, 240 Excretion 371 Explosives 450 f/number 77ff, 130 Fabry-Perot interferometer (see interferometer, Fabry-Perot) Falsification (of documents, art objects) 446 Fayalite (see QFM) Feldspar, K 325 Felgett advantage 100, 104, 124 Fiber monomode 130ff, 217ff multimode 130, 228 optical 217ff Fiber optics 124, 128ff graded optics 64, 128ff probe, indirect coupled 141 sensor, direct-coupled 131 Fibers 279ff Field emission 202 Figure of merit (of a laser beam) 56ff Filament 279, 284 Films island 399 Langmuir-Blogett 234ff, 240 Filter 102ff analog 161, 166 antialiasing 166 Bragg diffraction 103ff digital 166ff edge 102 holographic 104ff, 144 holographic notch 178ff interference 102ff notch lOOff, 144, 218 spatial 40ff spectral 177ff tunable acoustico-optic 180 Fluid inclusions (see inclusions, fluid) Fluorescence 20, 219, 224, 291, 326, 367, 375 minimization of 121 Fluorescent samples 121ff Focal length 202 Focal cylinder 28 region 28 Fore monochromator 102

Force constants 10 Forensic science 442 Forsterite 305ff, 440 Fourier transform (see FT) Fraunhofer diffraction 53 Frescoes 430, 437 Friend cells 405 Front lens 69 FT 155ff Raman spectroscopy 123ff, 127ff, 260ff, 279, 301 FT-SERS (see SERS, FT) FWHM 83, 146, 255, 265, 284, 335 Garnet 299, 302, 306 majorite 297 Gases, condensed 316ff Gaussian beam focusing of 52ff lens relation for 53ff Gaussian function 156 Gems 438 Ge02 316 Geochemistry 320ff Geological materials 290ff at high-pressure 30Iff Geology 224 Geometrical extent 81, 89, 100 Germanates 306 Gibb's ears 99 Glasses 343ff, 351ff Glycolipids 412 Glycoprotein 412 ai-acid 414 Graded index fiber optics 64, 129 lens 128 Graphite 312ff, 339 Grating 87ff, 102 holographic 87ff volume phase 180 GRIN (see graded index, lens) Group frequencies lOff Group intensities (Raman) llff Gun, electron 202, 225ff Gypsum 300 H2S 338 Halite 323ff Hadamard transform 188ff Helmholtz-Lagrange invariant 82 Hematite 340, 431, 440

Index 459 High-Tc ceramics 252 Histological sections 224, 370ff Histology 369 Histone 385 Hologram, volume phase 87, 104 Hot pressing 250 HPLC {see chromatography, high-performance liquid) Hydrates, Raman spectra of 323 Hydrocarbons 321 Hydrodenitrogenation (HDN) 244 Hydrodesulfurization (HDS) 244 Hydrogen 316, 341ff Hydrosol 393ff, 405ff Ice {see also water) 316, 319 Illumination global 176, 190ff line 182, 190ff point 182 wide field 177 Illuminations {see manuscript) Ilmenite {see MgSi03) Image analog 209ff digital 209ff processing of 210 Imaging 175ff direct 176ff parallel 176ff PMT 120 series 182ff spectrograph 94 Impregnation 245 Impulse function 152ff in situ analysis 312, 347, 442 Incident light cone 29 Inclusions fluid 320ff hydrocarbon 325ff in gems 440 Index modulation 104ff Indicators, biological 371 Indicatrix, scattering 59 Indigo 438 Infrared spectroscopy, micro- 328, 347, 352 Instrumentation 5Iff Integrated optics {see waveguides, optical) Intensity of incident light 28 of Raman scattering 29ff profile 183ff, 187

Interface 258 Interferogram 98 Interferometer 95ff Fabry-Perot 95, 99ff Michelson 80, 97ff multiple wave 95, 99 two-wave 99 Internal field correction 333 Introphcine 397 Inversion operation {see symmetry operations) Invertebrates 370ff Ion beam 225ff image 226 microscope 225, 231 vibrations 323 Iron oxide 431, 440 Irradiance 52ff Island films {see films, island) isotope 224ff Isotropic bandshapes {see bandshapes) samples 28, 31 scattering 28 ISS 245 Jacquinot advantage 100, 123 stop 124 Jewelry 438 K562 cells {see cells, K562) Kidney stones 372 Kramers-Heisenberg formula 1, 21 Kyanite 293 Lake 437 Lambertian source 61 LAMMA 228ff Lapis lazuli 428 Laser radiation, rejection of lOOff Laser Ar+ 54, 326 beam quality {see beam quality) deflection 187 figure of merit {see figure of merit) Kr+ 326 Nd:YAG 54, 123, 127, 291 scanning 182ff sources 2, 52ff Lawsonite 295

460 Index Lead chromate 447 Lead white 437 Least squares polynomial smoothing, LSP {see data smoothing) Lens camera {see camera lenses) compensating {see compensating lenses) electromagnetic 202, 217, 226 electronic 202, 225 electrostatic 202, 225ff focusing of Gaussian beam 53ff front {see front lens) objectives {see objectives, lens) relation for a Gaussian beam {see Gaussian beam) Lenses, ocular {see ocular lenses) LiF 299 Light polarized 13ff scattering 5ff Light beam, Gaussian 52ff Light flux (Raman) 37 Light gathering power 81, 89 Light sources {see laser sources) Light tube 43 Line illumination {see illumination) Line scanning 232 Line shape 15Iff Living cell {see cell, living) LMS 228ff, 370 Loading, of catalysts 245ff, 249 Lorentzian 156, 162ff LPM (line pair per mm) 77 LPMS 228ff Luminescence 122 Lysosome 368, 371

Methyl violet 437 Mg2Si04 302 MgSiOa 302ff Microelectronics 258 Microscope objectives {see objectives, microscope) Microscope analytical 231, 371ff confocal 182 electron 202ff, 227, 231 Microspectrometer {see spectrometer, micro) Microthermometry 335ff Mineralogy 223 Minerals 449 phase diagrams of 293ff, 301 phase transitions in 310ff thermodynamic properties of 307ff Mirror objectives {see objectives, mirror) Mirror aspherical 72 ellipsoidal 75ff, 21 Iff optics 212ff parabolic 72, 215ff Mitoxantrone 383 MOLE 39, 177ff Molecular vibrations 8ff Molybdenum oxides 441 Monochromator 80, 87 dispersive 87 Morphology analysis of 280 dependence 246 MTF (Modulation transfer function) 71, 77 Multiplex advantage {see Felgett advantage) Muscovite 325 Mutual exclusion, rule of 9

Madder 437 Magnetic vector 6 Magnetite {see QFM) Magnification factor 38, 42, 45 Manuscripts 422, 427 binding of 437 illuminations in 427 Mass spectrometry 225ff Material sciences 223ff Melts 343ff, 351ff Metabolism 370 Metallurgy 224 Metalothionein 367, 375 Metals, toxic 367, 371, 375 Methane 319ff, 330ff

N.A. {see aperture, numerical) NaNOg 300 Nanophase 244 Naples yellow 426 (NH4)2S04 299 NIR excitation {see excitation, NIR) Nitrates 321ff Nitrogen 319ff, 340ff Nonbridging oxygen 247 Nuclear pores 402ff Nucleic acids 367 Nucleus (cell) 368, 374 Numerical aperture, N.A. {see Aperture, numerical) Nyquist frequency 160

Index 461 Objective 60ff annular 69 apochromatic 66 biological 66 Cassegrain 70, 213ff dry 66 immersion 65ff lens 63, 91 metallurgical 66ff microscope 57ff mirror 65ff wide angle 32 Ocular lenses 379, 391ff Olivines 305 Optical extent 37, 43 Optical fiber Raman spectra of 134, 149 sensors {see optrodes) Optical sectioning 40ff Optics collection 29, 39 coupling 37ff, 45 transfer 44 Optrodes 131, 144 Order-disorder transitions 312ff Orientation, molecular 267, 272, 282 Orpiment 422 Orthoclase 300 Orthovanadates 441 Oxalates, oxalic acid 437 Oxygen 342 Paintings {see easel paintings, wall paintings) Paints 447 Palaeo-fluids 338ff Papers, dyed 434, 437ff Pastels 437, 446 Peptide 300 Petrology 293ff Phase amorphous 293 aqueous {see aqueous phase) identification of 293, 299ff Phenakite 441 Phonon 307, 313, 317 Phosphates 322ff, 372ff Photodiode array 108, 116 Photomultiplier (PM) 209 tube {see PMT) Photon 3 optics 201ff, 368ff Phthalocyanines 446ff

Physisorption 248 Pigments, synthetic 425, 433 Plagioclase samples 296 Planetary sciences 289ff PM291 PMT, imaging 107 Point illumination {see illumination) Point source, isotropic 59 Polarizability 7ff, 333 Polarization 244, 283 crystals 16ff gases 15ff inverse 21 leakage 28 liquids 15ff of incident light 28ff Pollutants, atmospheric 301 Pollution 371ff Polyethyleneterephtalate (PET) 280 Polymers 279, 282, 447 Polymolybdates 441 Polymorphs 250ff crystalline 293ff Pore cells 371 Pores, nuclear {see nuclear pores) Portlandite {see Ca(OH)2) Porto notation 17 Poynting vector 6 Precursor 245 Prefilter lOOff Pressing 250 Profiles of absorption bands {see bandshapes) Promoter 244ff Propellants 450 Protective coating 257 Proteins 373 Prussian blue 434 Pupil 38, 46 dimension of 78 Pyrite 441 Pyrope 440 Pyroxene 441 Pyrrhotite 441 QFM 339 Quartz {see also QFM) 294ff, 306, 322, 339 Radial distribution, Gaussian 28, 35, 47 Raman bandshapes {see bandshapes) Raman effect {see scattering) mechanism 2ff, 20 resonance 8, 15, 20

462 Index Rayleigh diffusion 218 Rayleigh range 53ff RDSC {see differential scattering cross-section, Raman) Red compounds {see alizarin, hematite, iron oxide, madder, vermilion) Reflection, specular 85 Resolution axial 201, 207 lateral 201ff, 368ff mass 226 spatial 40, 379 spatial (of optical fibers) 138ff, 141, 147 Resolvance 84, 96 Resolving power axial 42 intrinsic 89 Resonance Raman 23Iff identification of pigments by 425 scattering 20ff Restoration (of art works) 437 Restrahlen region 18 Ruby 440ff Rutile 294ff, 312 Samples colored {see colored samples) 23Iff damage to, degradation of 54, 259, 327, 380, 385 geological 290ff natural 293ff shocked 296 synthetic 293ff Sampling function 152ff of analog signals 161 of art works 423 theorem 158ff Sapphire 299, 439ff Savitsky-Golay smoothing {see data smoothing) Scanning laser 182 confocal line 182 Scattered fight cone 29 Scattering cross-section {see differential scattering cross-section, Raman) Scattering Brifiouin {see BriUouin scattering) cone 29 geometry 34ff Raman Iff

Rayleigh Iff, 7, 20 elastic 1 inelastic 1 two-photon 3 classical light 3 tensor 5ff resonance-Raman 12ff Schwartzschild {see objectives, mirror) SEM 204, 299, 370 Semiconductors 258 Serpentine [see Ca(OH)2] SERS 232, 367, 374, 379ff, 392ff -active substrates 379, 387, 392 electrode probes 403 FT 400 microprobe 384 Shah function {see sampling function) Shot noise 100 Shtishovite 302ff Sialic acid 412 Sialyloligosaccharides 413 SiC 296ff Siderite 326 Sight, keenness of 368 Signal-to-noise ratio 165ff, 189ff Signal averaging 152, 165 filtering 152, 165ff processing 149, 165 smoothing 152, 169ff windowing 165ff Silicates 306, 315ff, 347ff Silicon 261, 276 nitride 250 on insulator (SOI), structure 270 Sillimanite 293 SIMS 224ff, 370ff Si02 {see also Shtishovite) 294, 304, 344ff Skeleton 373 Slit function 151ff Soft modes 307 Sofid angle 29, 43 Sources, laser {see laser sources) Spatial resolution {see resolution, spatial) Spectral analyzers, nondispersive 80 Spectrograph 80 dispersive 90 imaging {see imaging spectrograph) Spectrometer 37 micro 51 multichannel 125 single-channel 125

Index 463 Specular reflection {see reflection, specular) Spherical aberration 63, 9Iff correction of 67ff Spherocrystals 372ff Spinels 315 Splitting, of vibrational bands 5 Spread factor 187 SOUIDs 253 STEM 207ff Stokes spectrum 3ff, 17 Stonework 437 Strain 260, 275ff Stray light, rejection of 84ff, 106 Stress field 251 Sulfides 321 Sulfates 321ff Sulfidation 248 Sulfur 341 Superconductivity 228, 252 Symmetry operations 9 crystal 17

TEM 203ff, 294, 315ff, 370 Tensor scattering 12ff polarizability 13ff invariants 15ff, 3 Iff Thermal conversion 277 Thermometry, micro- {see microthermometry) Throughput {see also etendue) 81, 89, 123 Time-resolved Raman 292 Tin 339ff Ti02 294, 312 Tissue biological 206ff, 368ff, 379 conjunctive 372 Titanates 306ff Titanium oxide 431 TLC {see chromatography, thin-layer) TOP 225ff Toxic metals {see metals, toxic)

Transfer function 163 Tridymite 296 Tube lens 58 Tungsten 339ff Ultramarine 427, 434, 436 Uranium 339ff V2O5 300 Vacancies 246 Vermilion 431 Vertebrates 370ff Vibrations, molecular {see molecular vibrations) Vibron 317 Vickers hardness 251 Wadsleyite 302 Waist beam {see beam waist) image {see image waist) Wall paintings 430, 437 Water 319, 336ff Wave vector 17 Waveguides, optical 234ff Wavenumber 29 WD {see working distance) WDS 204ff White compounds {see lead white, titanium oxide) Wolframite 339 Working distance (WD) 44, 65, 145ff, 212ff, 290 X-ray diffraction 315ff XPS 245 Yellow compounds {see Naples yellow, orpiment) Zebrafish egg 404 Zircon 299, 441 Zirconia, partially stabilized (PSZ) 251 Zr02 307