Chemical microscopy - ACS Publications - American Chemical Society

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Anal. Chem. 1986, 58, 1926-1937

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Review: Chemical Microscopy Peter M. Cooke McCrone Research Institute, 2508 South Michigan Auenue, Chicago, Illinois 60616

This is a review of the publications in chemical microscopy from January 1984 through December 1985, primarily as documented in Chemical Abstracts. The review stresses advances in microscopical techniques, primarily using the polarized light microscope, and in instrumentation related to present or potential analytical methods. As in previous reviews, certain topics are excluded; for example, virtually all publications concerning transmission electron microscopy or scanning electron microscopy are omitted, as are publications of a more theoretical nature. The alphanumeric referencing scheme and major text divisions are the same as in recent reviews, for the readers’ convenience: sections range from A1 through Z1, A2 through 22, etc., and citations are referenced within a section by the rightmost one or two digits as in A1.9 or R2.6. References cited here were obtained from a computer search of Chemical Abstracts and a manual search of regularly abstracted, major microscope journals. A few books of general interest to the amateur and professional microscopist are included. I am aware that the survey is not complete and apologize if I have overlooked important papers. Microscopists and researchers are invited to send reprints of papers they believe should be included in future reviews on chemical microscopy. However, it is hoped where this review fails, it succeeds in providing useful and challenging information to the microscopist. Perhaps no other investigation so fully demonstrates the capabilities of polarized light microscopy, nor creates as much interest throughout the scientific community, as McCrone’s conclusion based on microscopical evidence that the “Shroud“ of Turin is, indeed, an artist’s paintin . No chemical microscopist, when confronted with the evijence, could conclude otherwise. Other analysts, using less direct methods of evaluation that seem to contradict the facts, have disagreed; this reflects the limitations of some very sophisticated equipment, and says something about the understanding of chemical microscopy within the scientific community. The polarized light microscope is misunderstood as a research instrument and often abused by scientists, and yet the field of chemical microscopy continues to grow. In 1984-1985 over 1300 students were taught applications and techniques of the polarized light microscope at the McCrone Research Institute alone. The publications featuring the microscope suggest the increased popularity of an instrument that has been used in research for 300 years. Al. Books-General Interest. Bradbury (A1.1) introduced a concise, practical, and well-organized introductory book on the use of the light microscope. Understanding the functions of the parts of the microscope is stressed, as well as basic concepts of diffraction, resolution, magnification, conjugate planes, lens aberrations, and illumination in An Introduction to the Optical Microscope. The second edition of Optical Mineralogy by Shelley (A1.2) is a good introductory text. Included are mineral descriptions, thin-section and grain-mount techniques, determinative tables, principles of crystallography and optical mineralogy, and the use of the universal-s e and spindle-stage techniques. Geologists especially woul benefit if an optical text included more than a few pages on the proper use of the microscope and different t es of illumination and image formation. 0 tical and lectron Microscopy, Volume 9, was edited by lfarer and Cosslett (A1.3). Three articles on electron microsco y are presented along with reviews titled Binocular Image-dearin Microscopes, Laser Microanalysis, and Instruments for 8ptical Microscope Image Analysis. Optical Microscopy of Materials was written by Haynes (A1.4). The book gives a simple and quantitative account of the theory and use of the microscope. He discusses image

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formation, objectives and eyepieces, illumination, photomicrography, and polarized light microscopy. Various methods for viewing, including opaque stop, phase contrast, and interference (two beam and multibeam) are covered. Haynes presents relevant applications illustrating the techniques as used in ceramics, polymer science, and metallurgy. Meyer’s (AI 5 ) laboratory reference book Microscopy on a Shoestring for Beekeepers and Naturalists covers the processes, preparation, and microscopical identification techniques for the naturalist. The naturalist will find many varied methods for the preparation and microscopical dissection of bees. The amatew microscopist will find suggestions on how to inexpensively make his own hot plate, embedding oven, hand and rocking microtomes, and centrifuge as well as dissecting and compound microscopes. Geologists and students are often in want of a good representative thin-section atlas that contains great photomicrographs. Such an atlas is presented by Adams, MacKenzie, and Guilford (A1.6) in An Atlas of Sedimentary Rocks Under the Microscope. The book contains superb photomicrogra hs with extended captions. Additional material is given, inclu$q classification tables and preparation and staining techniques for thin-section studies. Crystallographers will find new ideas from the presentation of stereo pairs in An Atlas of Hypersterograms of the Four Dimensional Crystal Classes by Whittaker (A1.7). All classes and symmetry operations are represented by computer-generated stereographic projection pairs. His “fourth dimension” is fully explained and the Hermann-Mauguin notation extended along with presentation of his new crystal systems. Ford (A1.8) illustrates the use of the simple microscope from an historical perspective in Single Lens. The works from Robert Hooke through Antony Van Leeuwenhoek to James Brown are emphasized through discussion of their discoveries, observations, and illustrations. The book demonstrates the amazing power the simple microscope provided for 2 centuries. In Discover the Invisible Grave (A1.9) has an ideal manual that aids the novice microscopist and naturalist in the microscopical discovery of living microorganisms. He discusses different types of illumination such as incident light, polarized light, brightfield, darkfield, oblique, Rheinberg, and interference, modulation, and phase contrast. Included are specimen preparation techniques and suggestions for better photomicrography. The book documents the world of living microorganisms with photomicrographs and discussion of feeding, reproduction, and defense mechanisms. The structure, unique microscopic textures, classification, and identification of liquid crystal phases have been detailed in Smectic Liquid Crystals, a book for liquid crystal workers by Gray and Goodby (A1.10). They recount the historical discovery of smectic types and their use today. Much useful information is provided including sections on the microscopical identification of specific phase types. B1. Articles of General Interest. The varied interests of microscopists and the broad applicability of microscopical techniques have been captured in an annotated set of abstracts from the 30th and 31st international Inter/Micro gathering of light and electron microscopists and published in the Microscope (B1.1) and (B1.2). Novel and improved approaches in applying the microscope to problem solving are reported in a variety of disciplines; forensic, pharmaceutical science, biology and microbiology, geology, chemistry, asbestos analysis, art conservation, and pure and applied research are but a few. New and improved instrumentation and techniques for sampling, preparing, and viewing specimens for examination have been pursued along with advances in photomacroand photomicrographic methods, and will be of interest to the amateur as well as professional microscopists. Worth men0 1986 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 58. NO. 9, AUGUST 1986 Pelw M. CMke Is a Research Microscopist and Insbuctw at the McCrone Research Institute. wlor to w m n g at the instnute M,. Cooke was Vbe-President of Universal Laboratories. Pittsburgh. PA, which specializes in ail aspects of asbestos analysis end abatement monitorino. Mr. Caoke ' IecBiYed his BBChelor of S&ce degree from the Univershy of Akron in 1980. majoring in biology and chemistry, and recently Ccompleted his Masters course work in @ology there. His major areas of interest. cin addtion lo teaching. include polilnation b~ OWY.mineralogy. and micropahntoicgy.

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tioning is the discussion on teaching of microscopy at various schools and institutions. A 14th Century Chinese glassworks site was discovered at Ekehen in December, 1982. Unearthed samples of glasses and melting pots were examined by optical microscopy, electron microscopy, IR spectra, microprobe, and chemical analysis. Investigations of Yi and Tu (BI.3)revealed that the technology of glass manufacture was much different than that of European countries and included the use of feldspars as a main resource, fluorspar as an opacifier and flux, and coke as fuel. The development of optical glasses, including the improvement in color correction of microscope objectives prior to 1880 and the development of borate, phosphate, and barium glasses by Abbe and Schott from 1880 to 1883 is discussed by Hofmann (81.4). The validation in measurements of liquid-liquid or liquid-vapor contact angles in cylindrical tubes was accomplished through the use of photomicrographs of the meniscus by Ross and Kornhrekke (B1.5). The photomicrographs could be used when the astigmatic distortion, caused by the tube acting as a cylindrical lens, was counteracted, and the deviation of the meniscus from a spherical surface was negligible. Niemeyer and Gavrilovic (81.6) presented the use of the light microscope and ion microprohe mass analyzer in the examination of contamination-induced adhesion failures of inks, lacquers, and coatings on metal cans in "What's Wrong with the Surface?" Chemical microscopy was reviewed in 1984 by Kentgen (B1.7). It covered December 1981 through November 1983. See also: 82.3, 02.2, Q1.1, R2.1, W1.7. C1. Optics. Kirk and Nyyssonen ( C 1 . l ) developed a monochromatic, waveguide model to predict the optical microscope images of thick-layer objects, illustrating the effects of line structure on optical images. Huard (CI.2) used an objective visibility technique for the classification of macroscopic surface defects in reflection and transmission systems of optical instruments, based on ohservation of the specimen against a light background. The effect of defects and the surface finish of refracting image-forming optical systems operating with incoherent illumination at visible wavelengths was related to performance in the image planes by Martin (C1.3). The standards for defect specification were much higher for localized defects, like scratches, than for distributed defects, such as scatter from imperfect polish or coating. Zielinska-Roboginska (C1.4) reviewed (14 refs) the phenomena of optical activity, linear dichroism, elliptical dichroism, and birefringence for X-rav frequencies durine . diffraction. The characterization of surfaces by polarized-light reflecC I S~.) . tometrv was reviewed (41 refs) bv Ohtsuka (.~ Optdacoustic Rpectroscopy. a t&d for the study of optical spertra of very transparent materials. was the suhject of a review hy Pntel (C/.fi). Kamenev and Troimkii ( C l . 7 ) altered mirrors in the Tolansky interferometer so that reflection hands of equal thickness formed hright lines on a dark hackground in studies of opaque surfares. While studying fibers of thick. sheared films of liquid crysmlline polymers viewed between crnssed polarizers, Viney ( C I . 8 )discovered that enhanced resolution relative to axial Light is not alwavv achieved by the use of unidirertional oblique illumination. No improvement in resolution wns obtained hy filtering the incident henm when the conoscopir image of a

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sample contained only first-order maxima or first and higher odd-order maxima. Kothiyal and Delisle (CI.9) discussed inaccuracies and misinterpretations in the error analysis of azimuth angles and retardation of the Y plates used inside a rotating analyzer heterodyne interferometer. Phase measurements up to second order were affected. An automatic focusing system with an accuracy of 0.1 r m was employed by Xu (CI.10) in the study of light energy on the focal plane in optical projection microphotolithography. See also: B1.1, BI.2, 81.3, N1.6. D1. Instruments. A minifurnace equipped with a stereomicroscope for continuous viewing of single crystals grown from a melt was introduced by Forslund and Jelinski (D1.I). Aleksandrov and co-workers (01.2) described an automated scanning microscope that can operate in optical reflection, photo-, thermo-, or photoacoustic detection modes. Olympus Optical Co. (01.3)introduced an ultrasonic microscope with improved signal-to-noise ratio whose spherical lens portion contacted the sound-field medium of an ultrasonic focusing lens with a chalcogenide glass fiber acoustic impedance layer. Tanasugarn and eo-workers (D1.4) using an image processing system interfaced with a microspectrofluorometer obtained pH measurements in living cells. The TRE i-line 800 SLR wafer stepper equipped with Zeiss components for use in submicrometer optical lithography was introduced by Lee, Grillo, and Miller (D1.5). Chang (DI.6) evaluated the Ultratech Stepper Model lo00 with 1-pn lens and KLS automatic reticle inspection system. A Schlieren microscope was introduced by Prast (01.7) and was used to measure profiles of optical quality surfaces with accuracies of *10 nm for shape, f l nm for roughness, and with a horizontal resolution of f 3 nm. See also: BI.1, B1.2, N1.2, 0 1 . 1 , 01.4, VI.1, Q.2.

MICROSCOPICAL METHODS El. Polarized Light Microscopy. The polarized light microscope was featured as the primary investigative instrument in the study and classification of drugs from the Japanese Pharmacopeia X by Watanabe, Yamaoka, Kuroda, Yokoyama and Umeda. An immersion method was employed measuring morphological characteristics, refractive indices, and birefringence, A graphic representation of the correlation between refractive indices and logarithms of birefringence was recorded for analysis and identification. Thirty-two antibiotic drugs were recorded in Volume V (E1.1).Fifty-six drugs acting on the nervous system were investigated in Volume VI (E1.2), and seventy-seven organic crystallive drugs were examined for Volume VI1 (E1.3). Jones (E1.4) investigated the aggregation phenomena of water-based magnetic liquids using a phase compensator fitted on a polarized light microscope. See also: AI.1-1.5, BI.1, BIZ, C I S , 02.3, E2.2-2.4, E2.82.9, E2.1, E2.12, F2.1, F2.8-2.9, L2.2, N2.1-2.3, N2.6, 02.2, P2.1, R2.3-2.4, S2.3, T2.2, T2.4, V2.3. F1. Microphotometry a n d Microspectrophotometry. Achilles (F1.1) developed a method using a microscopephotometer and inverted microscope for determining relative rates of crystallization during Ca oxalate formation while utilizing gel crystallization. The method could he automated and enhanced by image analysis. Workable laboratory techniques written by Miksche and Dhillon (F1.2) cover the use of the microspectrophotometer, cell and tissue preparation, staining, development of internal standards, and absorption measurements. Morliere and co-workers (F1.3)studied the lysosomal enzyme release of single cells using microspectrofluorometry. Romanowsky-Giemsa staining using mouse fibroblast LM cells was studied by Naviak, Seiffert, Wittekind. and Zimmerman (F1.4). Ashmun, Hultguist, and Schultz (F1.5) quantitated glucose-6-~hosohatedehvdroeenase in sinele cells usine mi" crospectrophotometry: The distribution of metal-hearing amoebocytes in various tissues of the Pacific oyster Crossostrea gigas was examined by Lytton, Eastgate, and Ashholt (F1.6) using automated scanning microphotometry. A computerized microspectrophotometer that provided accurate, repeatable measurements of reflected light and fluorescence from cells, including nuclear DNA content, WBS 0

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ANALYTICAL CHEMISTRY, VOL. 58, NO. 9, AUGUST 1986

developed by Neely, Townsend, and Combs (F1.7).Fiber optic bundles in the light path of the microscope were used providing easy alignment, uniformity of illumination, and decrease of equipment size. Burr and Harosi (F1.8)used a dichroic microspectrophotometer for the in situ measurement of isotropic and dichroic absorbance in naturally crystalline hemoglobin of the nematode Mermis nigrescens. Groeger (F1.9)utilized IR and UV microspectrophotometry in the examination of polyethylene cable insulation. The construction of the UMSP-5 microscope-spectrophotometer for testing semiconductor materials was presented by Baurschmidt and Scharf (F1.10). Papayan and Agroskin (F1.I 1) developed a polarization microspectrophotometerfor simultaneous measurement of the coefficients of transmission and reflectance of microcrystals in the ultraviolet-visible spectrum. Prikryl, Ruzicka, and Burgert (F1.12)used an Opton 01type microscope-photometer in developing an accurate method of measuring concentration fields of dyes in synthetic fibers. Reflectance microspectrophotometry was employed to differentiate ink marks on documents by Clement (F1,13);he used reflectance curves in the 400-600 nm range to date ink marks or detect fraud. A near-IR, double beam, light-detection system was presented for use with a microspectrophotometer in polarized absorption spectroscopic investigation of single crystals by Zelano and Co-workers (F1.14). A new computer-controlled microscopical IR spectroscopy technique developed by Brenner (F1.15)was used to investigate areas of individual coal macerals as small as 20 pm across and makes possible individual functional group analysis of the macerals. See also: R2.6. G1. IR, UV, and Raman Microscopy. Elliott, Regnault, and Wakefield (G1.1) presented three modes of polarized IR microscopy: stress birefringence; absorption; and a unique, 1-pm-resolutiontransmission photoluminescence mode. They examined growth structures of liquid-encapsulatedCmhrolski indium phosphide and some related compounds. Addar, Boyer, Oswalt, and Nguyen ((31.2) discuss the use of Raman spectroscopy in the microanalysis of industrial samples, such as electronic materials, polymers, glass, catalysts, graphite fibers, and metallurgical materials. Manson, Ramirez, and Hertzberg (G1.3) used an IR microscope in the examination of fatigue in polymers. Coherent anti-Stokes Raman-scattering methods were explored for imaging biological compounds in microscopic samples by Duncan, Reintjes, and Manuccia (G1.4)and provided excellent molecular discrimination, high spatial resolution, and digital processing of images not found with any other current imaging technique. Truchet (G1.5) made use of Raman microspectrometry for the determination of cell and tissue composition in fish, spiders, insects, and mollusks. The laser Raman spectra for hemato orphyrin derivatives, pure and in solution, was investigated y Liu and co-workers (G1.6). De Mul, Otto, and Greve (G1.7) applied normal Raman and surface-enhanced Raman spectroscopy to study intact chromosomes and constituent compounds, like DNA bases. Herres and Zachmann (G1.8) gave examples of different techniques for the characterization of coatings and thin films using IR microscopy, photoacoustic detection, and differences in reflection with’ FT-IR. Microscopic structures of thin films of phosphorus were examined using Raman scattering by Olego, Baumann, and Schachter (G1.9). Kalikov and Markov (Gl.10) discussed the main standardization criteria in the laser microscopical analysis of minerals. Internal structures and textures in ore minerals cannot, ordinarily, be detected with reflected illumination. Campbell and associates ( G l . l 1) looked a t liquid-to-vapor ratios, daughter minerals, and liquid C02in growth banding and fluid inclusions utilizing IR microscopy, which permitted the measurement of homogenization and melting temperatures. Cheilletz and co-workers (G1.12) investigated the molar fractions of H,O and CO2/CH4from molecular fluids in a

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hydrothermal quartz vein, using Raman spectroscopy. Fluid inclusions of solid hydrogen sulfide and solid carbon dioxide occurring in minerals were identified using Raman microspectrometry by Dubessy and co-workers (GI .13). Nakamizo and Tamai (G1.14) examined oxidized and polished surfaces of glassy carbon using Raman spectroscopy. In situ analysis of surface f i i s on lead and asbestos-cement pipes was accomplished by Brown (G1.15)using laser-excited Raman spectroscopy and IR absorption. Hogarth and Hosseini looked at IR absorption in vanadate glasses (G1.16);Kugel and co-workers (G1.17)studied potassium niobate and Houde and associates (G1.18)studied @-lithiumiodate using Raman spectroscopy. Kassahma, Aoki, and Tatsuzaki (G1.19)studied the phase transitions in cesium dihydrogen phosphate and cesium dideuterium phosphate. The characterization of solids using Raman spectroscopy was reviewed by Nakeshima and Mitsuishi (G1.20). See also: 62.5. H1. Fluorescence Microscopy. A fluorescence microscope was modified to chop rapidly between two excitation wavelengths in recording cytosolic free Ca2+by Tsien, Rink, and Poenie (H1.1). Quackenbush (HI-2)incorporated ultraviolet fluorescence in the analysis of resins and coatings of paper. Lanni, Waggoner, and Taylor (H1.3)applied total internal reflection fluorescence microscopy in studies of the laminar organization of 3T3 fibroblast cells grown on glass slides. A system presented by Geel, Smith, Nicolaissen, and Winefordner (H1.4)used a pulsed laser source for quantitative epifluorescence measurements. This had advantages over systems using a continuous spectral source in examining low intensity level samples. Loesche and Moehwald (H1.5) describe an experimental setup to examine dynamic processes in surfactant monolayers at the air/water interface using fluorescence microscopy. Epifluorescence microscopy was used by Moroz and Kobilinsky (HI .6) to examine the fluorescence properties of quinine in studies of lung tissue sections. Luminescent and light microscopical histochemical methods were discussed by Gordon (H1.7). Yanagida (H1.8) reviewed (5 refs) video fluorescence microscopy. Cox (H1.9) introduced a scanning optical fluorescence microscope with improved resolution, among other improvements, that can be modified for automated fluorescence examination. Ultraviolet fluorescence microsco y was used to examine longitudinal ground-sections of teeti for the distribution of tetracycline by Nalbandian, Hagopian, and Patters (H1.10). A review (37 refs) of time-resolved fluorescence microscopy applied to biology was written by Docchio, Ramponi, Sacchi, Bottiroli, and Freitas (H1.11). See also: 12.5,S2.2. 11. Laser and Holographic Microscopy. Hirako and co-workers (11.1)developed a laser microscope system for use in photobiological and photomedical investigations. Applying laser microscope techniques to UV absorption, Tashiro, Minoh, Tsukakoshi, and Kasuya (11.2)obtained micrometer resolution using a small spot scanning method. Ambar, Aoki, Takai, and Asakura (11.3)discussed mechanisms of speckle reduction in laser-microscope images using a rotating optical fiber. Photothermal refraction for scanning laser microscopy was reviewed (9 ref) by Burgi, Nolan, Risfelt, and Dovichi (11.4). Zemskov, Kazaryan, Matveev, and Petrash (11.5) employed a laser projection microscope-oscillator-amplifier system to obtain sharp negative images at 510.5 nm and 578.2 nm of a Cu atom, by locating the intermediate image plane inside the active medium. A scanning optical fiber microscope produced laser beam induced current images of defects in polycrystalline Si solar cells for Ogura, Sakaue, and Tokumaru (11.6). It was equippd with a digital image processor and an optical fiber-positioning system. See also: 12.5,J1.8, J1.9.

ANALYTICAL CHEMISTRY, VOL. 58, NO. 9, AUGUST 1986

Jl. Interference Microscopy. Interference microscopy was used to measure liquid-liquid-solid contact angles in aqueous-organiegraphite by Callaghan, Fletcher, and Everett (Jl.1). Prentice and Hashemi (J1.2) compared Nomarski doublebeam interference contrast microscopy using transmitted and reflected liiht to other microscopical techniques in the study of polymeric materials. Gaudig and Scheck (J1.3)described a procedure for sample preparation using ion beam polishing for interference layer microscopy. To study cell types in mouse brain, the optical path difference in eu- and heterochromatin in karyoplasm was determined by Korr (J1.4) using interference microscopy. Chertkov and Chalykh (J1.5)described a method to study interdiffusion of polybutadiene and dioctyl phthalate using a polarization interference microscope. A photoelectric interference measuring microscope usin a He-Ne laser light source was incorporated by Zhou (JI.67 in observations of phase boundaries, crystal structures, integrated-circuit structures, and photomasks. Beier and Schollmeyer (J1.7) described a method for the characterization of high polymer fibers by interference holographic microscopy using a He-Ne laser. A new multiple-frequenc laser interference microscope consisting of a Mach-Zehndrer interference microscope and coherent laser light containing more than one lager line was described by Pearce (J1.8). The n&ow shift mode permitted measurements of frin e thickness and shift to exact calculations of refractive in&ces to