Planar chromatography - ACS Publications - American Chemical Society

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Anal. Chem. IQQO, 62, 423 R-441 R

Chemical Microscopy Peter M.Cooke McCrone Research Institute, 2820 South Michigan Auenue, Chicago, Illinois 60616

1989 marked the 150th anniversary of the founding of what is now the Royal Microscopical Society. In the last century and a half, the status of the microscope, which was once considered little more than an optical toy by the layperson, has been elevated to that of a scientific o tical instrument. Further, 1989 was heralded as the “Year o?The Microscope”. In reviewing the literature, it is no wonder that the microscope has achieved such a unique position within the scientific community as the 20th century symbol of science; no other scientific instrument affords both the amateur and the seasoned professional such equal accessibility. This review includes a few articles of historical interest from the many journals that joined in celebrating the “Year of The Microscope”. Classical references in microscopy are also included, as are critical evaluations of Anton Van Leeuwenhoek’s simple microscope and the first recordd microscopical observation in the literature. In stellar fashion, even a constellation is featured-”Microscopium”. The development of classical imaging techniques and their influence on microscopy today were reviewed while others compared the impact that current advances in image enhancement will have on the future of light microscopy. While the historical image of the microsco e was widely celebrated in 1989, the present-day image of tEe microscope and the microscopist was also evaluated at len h. Although the microscope is most often associated wit biology and medicine, lesser known applications that span many other disciplines are delineated in this review. New applications continue to emerge with advancements in improved imaging techniques and instrumentation. Yet, the field of microscopy is still misunderstood, and, with the advent of many high-tech instruments, the credibility of the light microscope as an analytical instrument is frequently looked. A number of res ected microscopists published articles reaffirming the credgility of the light microscope in particular and microscopy in eneral. Rochow maintains that microscopy is a technology to %elearned and not merely a technique. In terms of educating future microsco ists, McCrone asserts that “Microscopy is more a way of thiniing than a way of looking beginning microscopists need to do a great deal of lookin before they can think microsco icall . He has also prove8 that the light microscope a n i estaGished microchemical techniques still have unique applications when identifyin iron earth, vermilion, and titanium white pigments from the “win Shroud and Vinland Map. What follows is a selective review of the literature offered in chemical microscopy from approximately December 1987 through December 1989, with emphasis on progress in microscopy and microscopical techniques, and instrumentation devoted to present or potential analytical methods. References were obtained from regularly abstracted, ma’or microscope journals and from Chemical Abstracts. The skeer volume of references in 1988 and 1989 demonstrates a substantial increase in the continued use and development of relatively newer techniques, including video enhancement, confocal microscopy, acoustic microscopy, and near-field microscopy. A number of articles and books of general interest to both the amateur and professional microscopist are also included. Several new journals made their debut in the last three ears. Microscopy and Analysis, ublished bimonthly in the bnited Kingdom, successfully bri&es the ap between formal scientific journals and trade magazines. h a n y articles from its f i t 14 issues warranted inclusion in this review. The new quarterly, Journal of Computer-Assisted Microscopy, encourages.submissions on virtually every aspect of computer use in microscopy. This review is not intended to be a complete synopsis of the microscopical literature nor could it be,space restrictions alone do not permit it. Electron microscopy is the subject of

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a separate review; included are a few references to scanning tunneling microscopy. Publications of a more theoretical nature are also omitted. I am aware that the survey is not complete and apologize for any omissions of important work. Readers are invited to send papers (or suggestions) the believe should be included in future reviews on chemicdmicroscopy. Requirements necessitated a change in the referencing format from previous reviews. I apologize if this causes any confusion or inconvenience to the reader. Books of General Interest. Aduanced Light Microscop Vol. 1Principles and Basic Properties and Aduanced LigZ Microscopy; Vol. 2 Specialized Methods are the first two volumes in a three-volume work from the celebrated physicist, Pluta (I), who believes that “the microscope is an instrument that encompasses and successfully displays what are perhaps the most fascinating phenomena of physical o tics, namely, the diffraction, interference, and polarization of {ght. Volume 1is intended to provide the professional and the postgraduate study with the necessar background of classical light microscopy, as well as mo ern methods, techniques, and procedures of light microscopy. Volume 2 discusses various contrast techniques, emphasizing qualitative and descriptive methods. Reviewers claim it will be a classic. Principles and Practice of Microscopy and Scientific Photography is an excellent compilation of articles published by Klosevych (2) between 1984 and 1989 in the Bulletin of the Microscopical Society of Canada. The author succeeds in sharing his expertise in microscopy, photomicrography,and the production of audiovisual presentations. Photomacrography: An Introduction (3) is a book for the ex erienced pho apher; for the purpose of writing the book, “pKotomacrograp y” is defined as the magnification of 3:l through 1351. Chapters focus on the characteristicsand range of the human eye, photomacrographic systems, illumination and practical problems, methods and applications, hi h s and instant photomacrography, and techni ues in u\l&%d Photoatlas of Inclusions in Gemstones ?4) is a geautif y illustrated altas with over 1400 remarkable color photomacrographs taken under brightfield, polarized li ht, and darkprovides field conditions. Scientific in its coverage, the information on the use of inclusions to differentiate natural from synthetic emstones, formation mechanics of both mineral and flud inclusions, and information on the paragenesis of gemstones. A well-produced book (5)for both the specialist or amateur microscopist is Atlas of Dinoflagellates; A Scanning Electron Microscope Survey. Basic Histochemistry (6) is recommended to the analytical microscopist. Of special interest is the chapter devoted to the interpretation and assessment of histochemical resolution, which attempts to detail all the factors that can influence histochemical tests. A Guide to Materials Characterization and Chemical Analysis (7)was written to provide both novice and student interested in the characterization of materials with an analysis of chemicals, polymers, ceramics,metals, and composites. The book describes the basic theory, instrumentation, and applications of most analytical instruments used today. Each chapter is neatly divided into techniques and includes subdivisions that cover what the techni ue does, materials that can be studied and sample size, how &e technique works, and specific applications. Modern Food Microbiology (8)is a valuable reference for food microscopists, microbiologists, and patholo ish. Advances in Optical and Electron Microscopy, $01.10 has been edited by Barer and Cosslett (9) in keeping with the series format. This fine volume includes self-contained chapters on different subjects, each by a different author complete with references. It covers scanning optical mi-

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crosco y, holographic microscopy, photoelectron imaging, photoepyectron microscopy, and other techmques, plus a Wyear review of the development of instrumentation for the electron microscope. Practical Pedology Studying Soils in the Field (10)is one of the best introductorv books on the subiect. Manv of the practical methods and-techniques will be“of use to i h e microscopist in forensics and archaeology as well as to those in geological, environmental, or soil sciences. Introduction to Fluorescence Microscopy (11) has been specifically recommended for beginning university students, yet it is also valuable to the novice looking for a practical rather than theoretical approach to fluorescence microscopy. Composite Materials Handbook (12)provides the microscopist encountering new composites with much-needed background information on fiber- and metal-reinforced matrix composites. The Camera Lucida in Art and Science (13)has been called the definitive work on the sub’ed. The book details the early history from William H de dollaston’s invention. Microscopists will be delightegby Part 2, which is devoted to the camera lucida and the microscope. Of interest to the art conservator is The National Gallery Technical Bulletin (14)which provides an inside look into the site that some feel is the foremost laboratory for art authentication, conservation,and restoration. In addition to the annual s u m m a r y of paintings cleaned and restored during the previous year, Vol. 11contains six articles including ‘Analysis of Paint Media”. Image Analysis: Principles and Practice (15)offers 13 chapters covering acquisition, processing, detection, measurement, and analysis of images. While not a textbook, this technical handbook is extremely eas to read and will ap al to those seeking both an update an8a better understangg of new equi ment. Botanica Microscopy 1985 is a useful volume; 8 of 14 chapters deal with microscopy or microtechniques. HeslopHarrison (16)provides an exceptional compendium of botanical microscopy in Chapter 1, noting that recent techniques in microscopy and microtechnique now enable the microscopist to view rxceptional images of the living cell other than with an electroi beam. Asbestos and Other Fibrous Materials (17)provides an account of the mineralogy and uses of known asbestiform minerals, and discusses diseases associated with exposure to fibers. Of particular interest to the microscopist is the description of the asbestos mineralogy and complete crystal chemistry of fiber-forming silicate and aluminosilicate minerals. Artist’s Pigments; A Handbook of Their Uses and Characteristics was edited by Feller (18). The pigments included in the volume are indian yellow, cobalt yellow, barium sulfate, cadmium ellows, oranges and reds, red lead and minium, green eartg, zinc white, chrome yellow, and other chromate pigments, and lead antimonate yellow and carmine. Appendicized are light microscopical procedures used in the examination of the particles, terminology descriptions, and standard specifications of pigment composition and color. Taylor (19)penned the excellent and highly acclaimed Diffraction. It is recommended for all those microscopists who want an understandable, nonmathematical description of image formation and the information that can be obtained from an image. The classical microchemical procedures developed by Behrens and refined by Chamot (20)are reprinted in Handbook of Chemical Microscopy, Vol. II. This is the only available inorganic microchemical (microcrystal) textbook in print and is a must for all practicing microscopists. The Microscopical Characters of Artificial Inorganic Solid Substances: 0 tical Properties of Artificial Minerals by Winchell and d c h e l l ( 2 1 )is now in reprint. It covers optical and morphological crystal data for many minerals and inorganic compounds. The second edition of The 0 tical Pro erties of Organic Compounds by Winchell and kinchell 6 2 ) contains crystallogra hy, optical, morphological, and X-ra data (complete to 1952Pof 2500 common organic compoungs. It is the only source of crystallographic data on organic compounds. Wang’s (23)Crystal Optics of the 0 aque Minerals is now in second edition. Although written in Ehinese, it does contain

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32 pages of very useful detailed English abstracts. Vol. 51 in the Microscope Series (24)is a Glossary of Microscopical Terms and Definitions which contains more than 700 entries edited by a committee of the New York Microscopical Society. The updated edition of the Royal Microscopy Society Dictionary of Light Microscopy (25) makes recommendations for current usage and international standards as well as giving origins of microscopical terminology. Bradbury’s (26)revised edition of An Introduction to the Optical Microscope is highly recommended. The book stresses understanding the functional microscope parts as well as the basic concepts of diffraction, resolution, magnification, conjugate planes, lens aberrations, and illumination. Turner’s (27) The Great Age of the Microscope was published to commemorate the 150th anniversary of the found of the Royal Microscopical Society. The microscopes by the RMS are provided with complete descriptions, notes on the inventor or designer, and associated historical references to the scientific literature. Crystallography and Crystal Chemistry by Bloss (28) has been reprinted. The classic introductory text may be the best ever written on the subject and offers far more than an introduction. It is rife with information on many tropics: external symmetry; crystal classes, axes, and systems; crystal nomenclature and calculations; cr stal projections; crystal forms and class determination;tr&tion symmetry (latticea); internal symmetry (space groups); crystal chemistry; principles of crystal structures: structural variations, composition and stability; physical properties; crystals and light; and an introduction to X-ray crystallography. An Introduction to the Methods of Optical Crystallography by Bloss (29)has been reprinted and should find continued widespread use in introductory optical mineralogy courses. Articles of General Interest. Bracegirdle (30)wrote a concise yet detailed historical development and review of light microscopy from 1865 to 1985; articularly noteworthy are the many listings of classic regrences in microscopy and microscopical techniques. Ross (32)described some of his early research (1950s) in the development of quantitative techniques for studying living cells with phase contrast and interference microscopes. The descriptions on image formation and capabilities of the microscopes should be read b all racticing microbiologists. The recollections, many anceiotek: include his work with many famous microscope inventors, microscopists, and cell biologists. Ford (32)asserted the role of the simple microsco e in his reappraisal of the early history of microscopy and 6 3 ) documented the use of the Bancks dissecting microscope (ca. 1820). The early development of the reflecting microscope was detailed by Bradbury (34). Bracegirdle (35)provided an historical account of John Thomas Queckett and his interest in the practical microscopical observation of the healing process of wounds. The development and progress (36) of biological preparative techniques for light microscopy (1839-1989) were summarized by Bracegirdle. Emphasis is given to the introduction and establishment of 19th century techniques and the development of the microtome. Martin (37)discussed the history of dark ground illumination up to 1880, giving exam les of illumination both without special apparatus and wit! dry illuminators and immersed illuminators. The elevation of the microscope’s image in society from an optical toy to a scientific optical instrument was documented by Bennett (38). In a delightful article, Lane (39)emphasized the role of the microscope and microscopist in postage stamp production. From the start, ‘where the artist may use a microscope to produce the original design for a stamp, through the control of papermakin and printing rocesses to the philatelist or technician at tEe Post Office Juality Assurance Laboratory ..., the approval of the stamp is subject to the microscope”. The histor of users and specialist oups for microscopy in the Unitel Kingdom was reviewerby Claugher (40). A description of interests, contacts, and addresses was provided. The development of microscopical journals in the United Kingdom, United States and Canada, France, and Germany were reviewed by Brock (41).A s u m m a r y of the societies and publications role in scientific research is included.

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CHEMICAL MICROSCOPY Pdw M. Cooks is a R e ~ ~ a r CMicroscopist h and lnsbwta at h Mccrone Research Inst8ute. Wor to working at h inrfnuie. Mr. COOke was Vice-President of Universal Laboratorbs. Pmsburgh. PA. Mr. Cwke received his @achelor of Science degree from the Univershy of Akron in 1980. majoring in biology and chemistry. and recently completed his Masters course work in gsology there. His major areas of interest. in addition to teaching. include mineralagy. micropaleontolagy. and pollination biology.

Abramowitz (42) discussed the basic principles and importance of numerical aperture for microscopical image formation; a few of Abbe's experiments illustrating diffraction effects were presented. Every microscopist will be interested in Delly's (43) account of the common optical component in each and every microscopical observation-the human eye. The physical parameters that influence the resolving power, spectral sensitivity, astigmatism, and the sensitivity of direct and peripheral fields are presented. Problems and solutions in teaching introductory microscopical courses are discussed along with other phenomena such as mouches uolantes, dry eyes, amblyopia, strabismus, colorblindness, polarization, and flicker frequency: references to the microscopical literature are included. Bardell (44) provided an historical account of the first recorded microscopical observations in the Literature. He cites the observations of Federico Cesi and Francesco (1625-1630) on the bee and weevil. Elementary science teachers will benefit from Taylor's (45) continued work with the stereomicroscope in primary schools. His outline provides much guidance "in the hope that others may he encouraged to try using microscopes as part of a general science training program rather than just in support of biological investigations." Rappe (46) presented experiments for teaching introductory applied microscopy. The historical origins of the Royal Microscopical Society and its effect on the standards of scientific microscopy were recounted in the context of the times by Turner (47). The biographical account includes the Society's beginnings on September 3, 1839, and its acquisition of a library and microscopes through the iranting of a Royal Charter in 1866. Bradbury (48) provide insight into the sequence of events in the development of Microscopical Standards by the Royal Microscopical Society (1839-1939). The historical account neatly traces the standardization of both objective screw thread and gauges for eyepieces and substages. Cooke (49) provided a review (353 refs) of noteworthy articles on microscopy that were published from January 1986 through November 1987. The review is subdivided into seven categories: general interest; microscopical methods; sample preparation; examinations; applications; chemical microscopy: and analytical microscopy and microchemistry. Russ (50) introduced the new quarterly, Journal of Computer-Assisted Microscopy (Vol. 11,1989). Editon encourage papers on every aspect of computer use in microscopy. Topics range from "simulation of principles and instrument performance, control of instrumentation, presentation of information for human interaction, and interpretation of results. Deeply theoretical articles stand beside practical tips ... algorithms as well as images." Delly (51) traced the historical development of the capillary microscope and capillary microscopy: the light microscope currently aids in the capillary research of the skin, tongue, lip and gin iva, bulbar conjunctiva, and nailfold. Bracegirjle (52)reviewed the royal Microscopical Society's exhibit which highlighted 1989 as the year of the microscope. Agar (53)emphasized the need for and provided means of Calibration for magnification and resolution of light, scanning electron, transmission electron, and scanning microscopes; the requirements for a calibration specimen and traceable standards were included. McCrone (54) assessed the future of light microscopy by outlining past developments, including improved resolution, contrast enhancement, and microscopical technique, in ad-

dition to other advancements. He lauded modern innovations such as confocal illumination, video-enhanced imaging, and tandem scanning microscopy, yet cautioned that microscopists must still master basic microscopical technique to make full use of present technological advances. In another article, he continued by reviewing noteworthy microscopists and their achievements (55)and the present-day contributions that lead to his conclusion that there is a "renascence of light microscopy." The history of the littleknown constellation, Microscopium, was delightfully presented by Delly (56). Various historical cellestial maps illustrate the many graphical depictions of Microscopium. The bimonthly, Microscopy and Analysis (53,which debuted in September 1987, aims to provide microscopists in the United Kingdom (but, should find readership worldwide) with information on new techniques and products. It includes great color photographs, feature articles, information about references to microscopy and analysis in current literature, and new product descriptions. Abramowitz (58)reviewed the importance of, and outlined the steps in, achieving Kohler illumination. McCrone (59) reviewed the analytical findings that the Turin Shroud and Vinland Map are frauds: it is an excellent example that demonstrates the power and continued use of polarized light microscopy. A. Optics. Geometrical properties of the ray velocity surface were used by Viney (AI) in deriving optical resolution criteria for polydomain anisotropic materials. Pluta (AZ) reviewed achievements in light microscopy, gradient-n optics, optical processing via Fourier transforms, and classical and holographic interferometry at the Department of Physical Optics, Central Optical Labratory from 1983 to 1988. Singh and Singh (A31 reported the affect of third-order aberrations (spherical,coma, and astigmatism) on the intensity distribution in the images of a point object for the polarized light microscope with crossed polars. Nakamura et al. (A4) report that a new reconstruction algorithm using nonnegative constraints by a gradient-projection method dramatically improved the longitudinal resolution of tomographic imaging using an optical microscope. The concept and advantages of infinity color-corrected optics was presented by Keller (A5). Ratajczyk (A6) asks if the condition of the glass provided from manufacturers for optical instruments is of high enough quality. A quick, accurate, and precise method for calibrating microscopic laser beams is described by Stolpen et al. (A7). Klosevych (A8) reviewed the theory and stressed the importance of correct illumination [Kohler] and its role in providing optimal image formation: particular emphasis was put on the positioning of the lamp collector lens. Isaacson (A9) presented near-field scanning optical microscopy as a means of achieving spatial resolution rivaling the SEM. Fletcher (AIO) provided instructions and emphasized the need for visual inspection and detection of aberrations in microscope objectives by the star test. On-axis and off-axis aberrations were detailed. Lieberman et al. ( A l l ) put forth a method for the efficient emission of light from subwavelength dimensions based on packaging photons as molecular excitations and propagating them through micropipets that have inner diameters of 100 nm or less. The technique, on the cutting edge of optics technology, has the potential to dramatically increase the resolution of the light microscope. See also: 9, 19*24,25,27,28,30,32-34,37,42-44,50,014, G I , G2. GI. G2, G4. G4, I I ., 13-15.18. 13-15,18,JZ-J4.57. JZ-J4, 57, J9. J9, JIO. JIO, L4. L4, L6. L6, L7. L7, LIZ. LIZ, P7., Q1. P7.'Q1: 05. lfl.. U2. U2.'EE4 EE4. _ ~05: _ . ~1JI. B. Instruments. ('Iarke ( R I )descrihed the construction of an eyepiece auxiliary lens caps correctahle to the micros. copist's viiiiin. Delly and Sirovatka (R2)discussed the construction of a dedicated central-stop dispersion staining objective: a brief history OS con~merciallyavailable ohjectives was provided. Sirwatka WJdesigned a second dedicated central-stop dkpersiun staining gflhjective. I n a l a w article Sirovatka and Delly (B4J provided the development u i design features that enable modification of a nunadjustable, fixed-Sorusilluminamr of an Olynpua HHSP ANALYTICAL CHEMISTRY. VOL. 62. NO. 12. JUNE 15.

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illuminator to a focusable, fully adjustable illuminator capable of Kahler illumination. Construction details for a protective objective shield, t i d used when performin microchemical tests, was p r o v i g by drovatka and Delly d 5 ) . A self-leveling specimen holder for opaque, polished specimens was detailed by Love (B6). An im roved condenser system from Leitz which extends Kahler ilkmination to very low magnifications was described by Hmsch (B7). The system contains a light scrambler which provides stepless attenuation of light intensity ensuring uniform illumination of the objective exit pupil. An improved method for stereoscopic viewing with polarizing filters in a binocular microscope was postulated by Morrison (B8) based on the concept of Sculman. Winks (B9) provided instructions for a low-cost, easily constructed, transmitted light base for a stereomicrosco e using a fluorescence tube as the light source; an Olympus 8 Z stereomicroscope was adapted. The construction and use of a Mirau microinterferometer were set down by Loro (B10). The simple double-beam instrument mounts directly onto a metallographic ob’ective and reveals specimen contour or surface microtopograp y in white or monochromatic light. Wolf ( B l l ) introduced a beam-splitting microscope tube which allows Nomarski or phase-contrast stereo pairs to be taken with full resolving power on a single frame; epifluorescence was also considered. Maziarski (B12) delineated the instrumentation advances for inverted microscopy in addition to describing the development of the B & L Photo Zoom inverted microscope. Volume 243, Part 2, of Science (B13)contains an extensive ide to microsco es, optical equi ment, and merchant’saccessories; inclufd were acoustic,YJrighblight, cathode uminescence, combination li ht and electron, comparison, depth measuring, dissectin ckd-viewing, controlled atmosphere electron, micro robe e ectron, scannlng electron, transmission electron, fied’emission, fluorescence, forensic, hot-stage, infrared, interference, inverted, ion, laser-scanning, measuring, medical, metallurgical, modulation contrast, multiple-viewing, optical, phase, photomicrographic, pocket-size, polarizing, projection, research, scannin , acoustic, scanning tunneling, stereo, student, surgical, tifting stage, toolmaker, traveling, wide-field, and X-ray microscopes [sic]. Delly (B14) outlined how to convert dry objectives into immersion objectives for light microscopy. See also: 1,24,25,27, 30, 34,37,48-50,56, 58, B13, E2, E8,C5,13,15,K l , Lh,P7,Q1, Q5, U1, U2, U9,25,EE1, PP14, PP15, PP30, PP34.

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MICROSCOPICAL METHODS C. Polarized Light Microscopy. The year 1989 marked the centennial of the Michel-Levy interference color chart. Delly ( C l ) characterized the history, construction, and many uses of the chart, including rapid determination of thickness, birefringence, or retardation of microscopic samples viewed between fully crossed polarizers. Gunter (C2)advanced the use of the spindle stage and the computer program Excalibr in birefringence measurements of transparent material; measurements made visually or photometrically extended to fourth-decimal recision. The known relationship between the retardlation and the molecular o anization of collagen aided Whittaker et al. (C3) in studies oycerebral saccular aneurysms. The retardation measurements in Eosin- and Hematoxylin-stained sections were made with a Senarmont compensator. Ado1 hi and Schulze (C4)studied the formation of trans c r y s d h e structures a t polyethylene interfaces using a polarized light microscope. Watanabe and Ohnuma (C5) continued their pharmaceutical series with “A study of crystalline drugs by the polarizing microscope and x-ray analysis of the particle size distribution of henytoin”. aekeler (C6)described a microscopical method for early and rapid determination of chemically induced stress cracking of plastics which only requires small samples. Stress cracking using olarized light was most visible in amorphous versus partia ly crystalline polymers. The kinetics of the isothermal dehydration of single crystals of CuS04.5Hz0 was determined by analyzing mass-charge at

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various temperatures by Tanaka and Koga (C7); thin sections of single crystals were examined by polarized light microscopy. Hopple (C8)provided a review (9 refs) of reflected light fluorescence microscopy including a detailed description of the specific equipment and chemicals necessary to perform fluorescence microscopy with direct applications to lass fiber and graphite fiber reinforced epoxy composites an! polymer coatings on metals. Chemical microscopists will enjoy McCrone’s (C9)continued defense of the light microscope and the necessity of mastering microscopical techniques. He cites examples of scientific problems best solved with a polarized light microscope including forensic hair examinations, and fiber and pigment identification used in exposing textile and art forgeries. Most notable is his particle identification of iron earth and vermilion pigments leadin to his conclusion, su ported by chemical microscopists, &at the Turin Shrou! is a 14th century painting. See also: 1,19,20,24,25,28,30,50,54,55,58, A3, Bl-B4, B13, 0 1 1 , Fl, H l , I 3 , I 5 , Q l 1R l , U1, U l l , Y2, Y12, Y13, AA3, BBl-BB3, CC7, CC9, CClO,0 0 3 , 0 0 4 ,IIl, JJ4, JJ13, JJ17, KK1, M M 2 , 0 0 1 , PP4-PP6, PP8-PP10. D. Microphotometry and Microspectrophotometry. Ryan et al. (01)outlined basic features in the evolution of FT-IR microscopy and noted that increased use by microscopists dictates the need for instrumentation specifically designed for the microscopist. Applications of microspectroscop in the near-IR region were illustrated by Smith and Carl $2). Included were examples from the fields of materials science, single crystals, forensics, and biology. Infrared microspectroscopicsolutions to manufacturing and quality control problems were reviewed (8 refs) by Schiering ( 0 3 ) ; transmission and reflection measurements aided in detecting imperfections in polymers, contaminants on Si wafers, and imperfections on a magnetic disk. A microscope using a He-Ne laser source and microphotometer was developed by Devyat kh et al. ( 0 4 )to determine the concentration and size of fght-scattering centers in fluorozirconate glass. Reffner ( 0 5 )presented the design of an all-reflecting microscope for microspectroscopy. Reffner and Messerschqidt ( 0 6 )reported on the capabilitiesof lT-IR microspectrometry. An inte ated molecular spectrometer for microanalysiswas presentergby Ryan (07). Milledge and Mendelsohn (08) reviewed (13 refs) infrared microspectroscopy with special reference to computer-controlled mapping of inhomogenous specimens. Jouan et al. ( 0 9 ) determined the oxidation products in photooxidized matrixes of polymers by microspectrophotometry. Experimental studies by Zeichner et al. (010)revealed an inherent advantage of transmission microspectrophotometry of single-inked fibers versus the nondestructive transmission method due to large variations in the opacity of paper substrates and bronzing interferences. Humecki ( 0 1 1 ) made use of an FT-IR spectrometer equipped with a microscope for the analysis of small samples of polymers containing contaminants or impurities. Polarlzed light was diverted thro h the large cassegrainian condenser lens of the microscope3y virtue of a mirror in the sample chamber. Several new and improved techniques for analyzing polymers based on FT-IR spectroscopy were presented by Compton (012). The technique allowed for the stud of single fibers, residual monomer levels, crystallinity a n i drawing properties, coating, curing reaction, impurities, and inclusions. The DNA content of human sperm was measured by an Opton-VMSP-30 type microspectrophotometer by Xue et al. (013). Boguth and Piller (014) summarized how the shape and size of the illumination aperture influenced the accuracy of microscopy-photometric measurements of internal transmittance. Microspectrophotometry was employed by Nearing (D15) in investigations into the structure and dynamics of the interstitial matrix of the rat mesentery. See also: 24,25,50,51,B13, S2, Y2, EE7, JJ4, JJ13, LL16, LL17.

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E. IR, UV,and Raman Microscopy. Billingham and Calvert ( E l )reviewed the instrumentation requirements for the ultraviolet microscopy of polymers. Bergin and Shurvell (E2) opened up new areas for Raman spectrosco b showing that near-IR excitation coupled with a modifief&& IR spectrometer circumvented the problems of laser-induced fluorescence. The two outlined some initial experiments on Fourier-transformRaman microscopy utilizing a conventional microscope. Examples of applying IR microimaging to organic coating on steel and latex-coated paper samples were sited by Carl (E3)the technique couples an IR microscope with a motorized x-y stage and offers information on thickness and coating variation. Schlotter (E4) reviewed (245 refs) the basics of Raman spectroscopy as applied to polymers. The review indicated current directions in research. Bowden and Dixon's (E5) reyiew (9 refs) examined experimental applications of Raman microscopy. Young (E6)made use of high-quality IR spectra on single filaments of Kevlar and Nomex aramid fibers, establishinga structure-property relationship with orientation. The desi n rinci les and performance of a second-generation, sinp!e-c\annep detector Hadamard Raman microprobe capable of diffraction-limited resolution were described by Morris and Treado ( E n . Gerber et al. (EB)obtained an image and IR spectrum using a scanning optical microscope. The microscope operated in conjunction with an IR detector and IR source. When compared to Nomarski microscopy, Raman microscopy showed increased sensitivity to detect subtle structural modifications of laser-induced damage in solids for Fauchet et al. (E9). Quantitative measurements of stress. strain. and crystailinity 'were also offered. See also: 24, 25, 50, 58, B3, N4, S2, U4, Y2, Y3, Y8, Y9, 23, BB1, BB5, EE?, "1,112, JJ15, JJ20, JJ21, LLl7, MMl, F. Fluorescence Microscopy. Results from microtubule dynamics in the chromosomal spindle fiber analyzed by fluorescence and hi h-resolution polarized light microscopy were recorded by Ckssimeris et al. ( F l ) . Mayfield and Munawar (F2) designed a system to operate with a UV fluorescencemicroscope that provided video images of the chlorophyll fluorescence of living algae cells. Miller (F3)characterized double-chain surfactants using video-enhanced microscow - _ and time-resolved fluorescence quenching. Delly (F4)outlined the apparatus and chemical formulations essential for the preparation and microscopical examination of phosphors, describing the kiln, ultraviolet light sources, reaction vessels, reagents, and balance needed. Descriptions of the nature of phosphors and photographs illustrating their characteristic luminescence were provided. A simple Langmuir trough allowing in situ observation of a monolayer on water over a wide area was designed and constructed by Miyano and Mori (F5). The trough can be fitted to a commercially available inverted fluorescence microscope. A novel fluorescence microscope configuration for real-time observation of Langmuir-Blodgett transfer was presented by Riegler (F6),enabling ima ing of any portion of the entire monolayer or water subp ase, substrate, or meniscus. Demian and Schwartz (F7)introduced a rapid method of calibrating fluorescence microsco es that allows the microscope photometer to be easil calitrated on a daily basis; the system is based on series of xi hly uniform cell-sized microbeads that carry different levefs of fluorochromes calibrated in equivalent soluble fluorescent molecules. An im roved fluorescence ap aratus from Schneider et al. (F8)appied the use of optical fikrs,offering the advantages of setup flexibility and improved quality of the illuminating beam for more precise measurements of correlation functions. Video-fluorescence microscopy was utilized by Harada and Yanagida (F9)to directly observe the sliding movement of single fluorescently labeled actin filaments along myosin fixed on a glass surface. Trout et al. (FlO)discussed the merits and weaknesses improving such an application. They also indicated future directions to be taken for element-s ecific epifluorescence microscopy when monitoring metafbiotransformations in environmental matrices.

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Caretta and Saibil (F11) introduced a new method for visualization of cyclic nucleotide binding sites in the vertebrate retina utilizing fluorescence microscopy. A multi-decay-time fluorescence ima ing apparatus was patented by Honig and Smith (F12). Ttey also put forth a method for resolving spatial differentiation and correlation of a plurality of separate sample components tagged with site-specific dyes. Moehwald (F13) reviewed (37 refs) the range of lateral structures detected in ultrathin organic films using newly developed techniques of fluorescence microscopy and X-ray scattering together with more conventional methods. Scheuerlein et al. (F14) presented an early quantitative method for measuring germination in nongreen spores of Pryopteris paleacea using an epifluorescence technique. Meller (F15) made use of and described an integrated comDuter-assisted video microscoDe for investieations of " mon'olayers on liquid and solid suhstrates. See also: 1 , 11,24,25,50,58,B13, C8, J8, J12, N 6 N 9 , Y5, Y7. CC2. CC4. CC6. KK3. LLl. LL6. LL11. LL14. G. Laser and Hdographic 1\;Licroscopy.'Block et al. (GI) utilized synthetic holograms for high-resolution scanning acoustic microscopy; sharper object edges and image structures more clearly related to the light microscopical image were reported. Calculations and measurements for the miniundulator device used as a radiation source for soft X-ray holography and scanning microscopy, which require high brightness for practical operation, were presented by Rarback et al. (G2). Basener (G3) described a technique that uses a combination laser, microscope, and mass spectrometer for analyzing defects in aint films. fiber-optical laser spot microscope allowing simultaneous measurements of photocurrent and reflected light intensity or the measurement of laser spot photocurrent under the illumination of other light sources was developed by Carlsson et al. (G4) to study semiconductor electrolyte interfaces. Armstrong and Fletcher (G5)m fied the vendor-supplied hardware for delivering the laser beam into the micro-Raman microscope, making beam delivery adjustments more userfriendly. Calibration of a krypton fluoride laser-plasma source for X-ra microscopical applications was put forth by Turcu et al. ( d 6 ) . Admixtures of hydrogen improved the uniformity of gain across the active media of pulsed copper bromide vapor lasers yielding brightness amplification in a layer projection microscope for Astadjob et al. (G7). It was possible to obtain quality amplified images several meters from the amplifier. Pileri (G8) developed a laser Doppler microscope and applied it to intracellular velocity profiles in real time. Crostack et al. (G9) reviewed (13 refs) the ultrasonic technique, acoustic microscopy, and interferometric holographic applications on failures adjacent to surfaces in platings and coatings. A nonlinear optical method capable of revealing surface roughness fractures about 1 nm in size was presented by Aktsi etrov et al. (GlO). Scanning tunneling microscopy estabished the limits of sensitivity. See also: 1,9,24,50,58,A?, B13, H5, KlO, L4,L5,M5, M6, M14, T5, EE3, LL5. H. Interference Microscopy. An apparatus for use with a polarizing interference microscope to determine the birefringence and mean refractive index of strained polymer fibers was designed by Hamza et al. ( H I ) . Kuzmin et al. (H2) applied a holographic interference microscopy technique that determined the overheating temperature of GaAs/AlGaAs and InGaAsP/lnP laser active layers at different pump levels in order to establish thermal resistance values. Foster (H3) outlined the theory, current instrumentation, and practical use of differential interference contrast in light microscopy. Included was a discussion on image interpretation of DIC microscopy. Quested and Bennett (H4) provided a basic outline of interference film microscopy that included theoretical considerations, methods of film deposition, and applications of the technique for identifying phases and quantifying structures. A new three-dimensional, noncontact laser interference microscope which uses computerized phase measurement in-

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terferomet to achieve subnanometer vertical resolution was developedyy Biegen and Smythe (H5). Shearing interference microscopical studies of fiber end-face topography of graded-indexand step-index optical waveguides were reported by Mangold and Schoeppe (H6). Bennett (H7) reviewed the concept of surface roughness and how to measure it by profilin and scattering methods. See also: 1 , 2 4 , 2 5 , 5 0 , 5 8 ,JlO, B13, E9, JI3, Q l ,Z 1 , 2 5 , DD1, JJ4. I. Phase Contrast and Schlieren Microscopy. Benwhop (11) successfully combined differential phase contrast and confocal microscopy by focusing the light of both pu il halves onto point detectors. The implementation of tlis detection scheme and its experimental results were enumerated. Weisen (12) presented the phase contrast method as an imaging diagnostic tool for plasma density fluctuations using a 23-cm-wide C02laser beam transmitted through the plasma, producing an image where small shifts due to refractive perturbations are revealed as corresponding intensity variations. The design, construction, and application of improved multisection polarizing filters to be used for variable asymmetrical contrast microscopy were put forth by Stran e (13). The redesigned filters can be used with objectives 4X tfrough l00X providing improved resolution and enhanced control over image contrast in producing pseudostereoscopic high contrast images of translucent and transparent phase specimens. Yu and Wei (14) used holographic phase-contrast photomicrogra hy to study the state of solution and the morphlogy of crystarsurfaces during growth. Conditions for improving the resolution and quality of the reconstructed image were described. Wilson (15) provides the means for enhancing differential phase contrast images by replacing the semicircular detector with a quadrant detector. Wilson (16) also evaluated the optical sectioning capability in relation to the shape and size of inhole/detector conformation. eouture (17)presented differential schlieren contrast for a reflected light microscope. Real-time and all-optical imaging techniques such as phase contrast, contrast improvement, and reversal were demonstrated and applied by Ngu en et al. (18) in explorin the possibilities offered by OpticJnonlinearities such as the err effect, nonlinear absorption, and stimulated scatterings. Lawrence and Birnie (19)presented a schlieren system and brief description of experiments demonstrating crystallization intended for earth science classes. Phase contrast matching was successfully ap lied on samples with the lowest molecular weight in lame1 ar structures composed of mixtures of labeled and unlabeled block coolymer for small-an le neutron scattering as reported by katsushita et al. (116. Differential phase contrast imaging in a scanning TEM was utilized b Morrison et al. (111)to measure very narrow domain w a d in the ferromagnetic alloy samarium-cobalt. Darkfeld and phase contrast yielded a resolution of 30-200 A in stud ing point defect clusters, dislocation loops, and helical diskations in Czochralski-grownNdYAG crystals for Deng et al. (112). Unfixed eyelashes and their follicles were described using dark ground and phase contrast microscopy by Hillman and Jarman (113). See also: 1, 24, 25, 31, 37, 50, 58, B3, B11, J13, Q l , U22, 25, PP33. J. Confocal Microscopy. Minsky (J1) provided insight into his investion of the confocal microscope over 30 years ago. Boyde (J2) rovided a review (28 refs) on confocal scanning microscopy. brief history was provided along with basic principles, surface images, extended focus images, stereo imaging, stereology, and a comparison of tandem scanning and confocal scanning microscopes and their applications. Explanations for image improvement in c o n f d microscopy were presented by Sheppard (J3). Shotton (54) presented both the advantages and the disadvantages of unitary beam confocal laser microscopy. Recent developments in confocal imaging, superresolutiontechniques, and digital image processing and stereoscopic animation of confocal images were discussed, as were biological and medical applications.

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Becker (55)presented an introduction to tandem-scanning light microscopy highlighting the advantage of "noninvasive" methods. Wilson (J6) discussed the practical use of the scanning optical microscope noting examples of their application to the semiconductor industry. The advantages and disadvantages of on-axis and off-axis designs in confocal laser-scanning reflected and fluorescence microscopy were outlined by Draaijer and Haupt (J7). The two presented a real-time confocal laser scanning microscope suitable for reflection and fluorescence microscopy. Shorter frame times were achieved b scanning the laser focal oint with a fast acoustooptical de&ctor for line scanning antwith a scanning mirror for frame scanning. Takamatsu and Fujita (J8) examined chromosomes with a scanning laser confocal fluorescence microscope using image processing to create stereoscopic image pairs. Boyde (J9)discussed the use of transmitted light in addition to the normal reflection im ing mode in a tandem scanning reflected light microsco ;%e transmitted image is used to locate a field which is t K n examined in optical sections by the confocal mode. Carlini (JIO) illustrated the effects of using a misaligned or finite-sized detector on the various confocal imaging modes. Carlson and Liljeborg (J11) described a confocal laser microscope scanner for digital recording of optical serial sections. Shotton (J12) described the principle of confocal laser scanning fluorescence microscopy and provided examples of its use for obtainin precise noninvasive serial optical sections of fluorescently la eled biological specimens. The capabilities of a confocal scanning o tical microscope in materials microscopy, semiconductor fairure analysis, and biological microscopy were discussed by Dixon et al. (J13). Their work featured applications such as differential phase contrast, confocal sectioning, and optical beam-induced current imaging. Kino and Corle (J14)outlined the principles and applications of confocal scanning microscopy; the techniques and capabilities were compared to standard optical microscopes. Yatchmenoff (J15) reviewed (9 refs) the use of confocal scanning optical microscopy in evaluating the TSM-1 microscope. Various imaging modes of a confocal scanning laser microscope allowed Russ (J16)to measure internal structure and surface relief. Examples include biological specimens such as swimming paramedium and lant leaves as well as metalization structures in integratezcircuits and pores in highly dense A120,. See also: 55,58, B13, F 4 , I l , T5, T7, EE3,112, LLl, LL5LL7. K. Ultramicroscopy. Wickramasinghe (KI)provided insight into the capabilities and versatility of the relatively new family of microscopes including scanning tunneling (STM),magnetic force (MFM), and atomic force microscopy (MFM). Bonnell and Clarke (K2) provide a good introduction to scanning tunneling microsco y and spectroscopy, indicating applications for poorly congcting ceramic materials. A review (161 refs) of the achievements and construction of the scanning tunneling microscope was put forth by Kuk and Silverman (K3). The entire issue of Ultramicroscopy 25 (K4),99-181,1988, is devoted to scanning tunneling microscopy. It contains selected state-of-the-art papers 'ven at an evening workshop of the German Society of EM (&I. The entire issues of Parts 1,2, and 3 of the Journal of Microscopy, Vol. 152, Parts 1-3 (K4),were devoted to scanning tunneling microscopy. Duerig et al. (K5) evaluated tip sample interaction forces during normal tunneling operation using an Ir tip and a polycrystalline Ir sample. Scanning tunneling microscopy was included in a review of surface analysis by Si norelli et al. (K6);many other instruments were reviewel An e itaxially grown film of Au on a cleaved mica substrate yieldexan atomically flat surface allowing IR and scanning tunneling microscopical measurements on identical samples for Hallmark et al. (K7). The implications of obtaining high lateral/spatial reao!ution by placing a photon detector near the tip-sample region of

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a scanning tunneling microscope were discussed by Gimzewski et al. (KB). Isochromatic hotoemiasion spectra were measured of polycrystalline Ta an# Si (111)7 X 7 at photo energies of 9.5 eV. A laser ultramicroscopical method was developed for the determination of the concentration and size of light-scattering centers in a fluorozirconate glass by Devyatykh et al. (K9). Akahori (KlO)suggests that mechanical pencil leads 0.3 or 0.5 mm diameter between alligator clips can be electrically heated to eva orate carbon; a tun sten spiral sleeve serves as a source ofevaporated Au, Au--d, Pt-Pd. Safety in the electron microscope room was addressed by Chapman ( K l l ) . See also: 5 , 9 , 2 4 , 5 0 , 5 4 , 5 8 ,B13, G10, I l l , L3, M5, M15, N5, 01,P3, T4, U5, U18, Y12, BB3, EE14, K K l , LL4, PP13, PP32. L. X-ray Microscopy. Markowicz and Van Grieken ( L l ) reviewed the principles, instrumentation, and analytical aplications of X-ray microscopy, X-ray imaging, X-ray horescence, proton and particle induced X-ray emission, and electron microprobe microanalysis. In a review (34 refs) Shinozaki (L2)notes that much of the efforts in imaging with soft X-rays has been devoted to the development of ap aratus and methods, but with increased availability of soft ft-ray microscopes, much will be expected in applications to material science. Developments of the different types of X-ray microscopy were described in a review by Michette (L3). The images obtained were compared to electron and optical microscopy. A com act scanning soft X-ray microscope using a laserproducef plasma source and normal incidence mirrors was constructed by Trail and Byer (L4);the microscope operates a t a wavelength of 14 nm and has a spacial resolution of 0.5 nm. Richards et al. (L5)compared synchrotron and laser source images in X-ray contact microscopy of metal-contaminated biological tissue. Resolution of 70 nm was obtained with the synchrotron radiation source. Kirz’s (L6) review (28 refs) of X-ray microscopy included a brief survey of recent developments in instrumentation that provide greater speed, hi her resolution, better contrast, and additional information a out the specimen. Yamashita and Tsunemi (L7) reviewed (35 refs) recent ro ess of X-ray optical systems in the wavelength 1-300 A. k e g c t o r and detector X-ray characteristics for grazing and normal incidence imagin optical systems were included as well as discussion of multiayer reflectors, fresnel zone plates, and the use of charge couple devices as X-ray dectors. The otential application of transmission multilayer optics in the fevelopment of high-resolution X-ray microscopy and s ectrometer systems was characterized by Jankowski et al. (E8). Cheng (L9) presented advances in X-ray microscopy. The performance of collimated and focused synchronous radiation was compared by Jones et al. (LlO). A morphological study by Panessa-Warren et al. (L11) demonstrated a rapid, easy-to-use method of soft X-ray absorption ed e imaging for viewing biological specimens. The absorptive c\ara&ristics of monochromatic X-rays above and below the absorption edge of a specific element were exploited. An optical system based on the Talbot effect with modulated spatial coherence of radiation yielded high-resolution images for Aristov et al. (L12). A diffraction Talbot lens for ad g X-ray microscope was proposed, and the dispersion properties of the Talbot effect were analyzed. Wang (L13)introduced a method and microscope for investigating materials with X-rays. Optical components for X-ray microscopy and X-ray holograph were evaluated in an undulator radiation optical system gy Kakuchi et al. (L14). A grazing-incidence concave toroidal mirror was used by Hills et al. (L15)to relay soft X-ra s generated by a plasma source to the window of a wet-cell [older of hydrated, living cells. The algal material remained in a hydrated state during and after the imagin rocess. The device effectively reduced the debris generate! y a laser-plasma source. Turcu et al. (L16) detailed the calibration of a krypton fluoride laser-plasma source for X-ray microscopical applications.

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X-ray optical components, including fresnel zone plates and transmission gratings, were fabricated and evaluated in an undulator optical system by Kakuchi et al. (Ll7). Bateman et al. (L18) developed a microfocal X-ray generator combined with a multistep avalanche/multiwire roportional counter to produce a digital X-ray microscope. h e digital data sets obtained were employed to develop 3-D tomographical reconstruction techniques. A two-dimensional laser interferometric encoder for the scanning soft X-ray microscope was developed and tested by Shu et al. (L19). The instrument was designed to permit scanning speeds up to 3 mm/s and provide a resolution of 316 8, within the 3 mm range of the stage. Fresnel zone plate lenses with feature sizes as small as 50 nm were constructed and used in a scanning X-ray microscope by Vladimirsky et al. (L20). The biological images obtained highlighted protein enzymatic granules in an aquatic environment. The internal structures of intact insects were imaged b using rojection X-ray microscopy by Davies et al. (L21) a resoktion about 1pm. Information was obtained from live specimens, but dehydration by freeze-drying yielded more detailed images. See also: 24, 25, 50, 56, 58, B13, F14, G2, G l l , EE6. M. Acoustic Microscopy. The basic principles of acoustic microscopy and its applications in materials science to the study of ceramics, composites, pol mers, metals, and semiconductor materials were offered gy Issouckis ( M l ) . Fatkin et al. (M2)summarized the applications of acoustic microscopy to engineering ceramics and ceramic fiber composites, both low-ductility material. They also presented information on Raleigh velocity and attenuation used to characterize the microstructure of ceramics and ceramic composites. A review (4 refs) of scanning acoustic microscopy for metallographic examination in o rational, materials testing, and quality control was providegeby Dieser and Matthaei (M3). Khuri-Yakub (M4)reviewed (21 refs) the operation and use of acoustic microscopy in material characterization. Barthel (M5) summarized the experimental setup and fundamental processes of scanning acoustic microscopy, laser scanning microscopy, and scanning tunneling microscopy, emphasizing their importance for structural elucidation in material science. Ma and Zhang (M6)presented the operating principle and application of scanning light acoustic microscopy in detecting defects in ceramics. Experimental results in various ceramics indicate that high-frequency ultrasonic (100 MHz) can be used to detect defects smaller than 100 pm. A scanning acoustic microscope was built and its main features were described by Fossheim et al. (M7). A review (3 refs) was given by Kessler (M8)on the approach and application of scanning acoustic and C-mode scanning acoustic microscopy. Guenther et al. (M9)introduced a new method for imaging topographic features of nonconducting surfaces with a otential lateral resolution in the submicrometer range. #he scanning near-field acoustic microscopy has a distance sensor consisting of a sharply pointed vibrating tip, which is part of a high-Q quartz resonator driven at its resonance frequency. The decrease of the resonance frequency or amplitude of vibration when an object comes into proximity of the tip serves as the signal. A scanning laser acoustic microscope was used by Boehning and Tuohig (MlO)to evaluate the integrity of Mn-Zn ferrite ceramic components. Sherar and Foster ( M l l )constructed a scanning ultrasound microscope using a spherically focused PVDF tranducer. The system operated in 50-110 MHz and had a corresponding lateral limit of 17.5 pm. The design produced two imyem: attenuation images of thin specimens and darkfie d backscatter images of cross-sectional planes within specimens