(MP) Signor, A,, Biondi, L., Tamburro, A. M., BordiDon, E., Eur*J *B i O c h ~ . , 7,328 (1969). (6,iP) Siheimer, J. E., Hong, D. D., Stewart, J. T., Fink, M. L., Burckhalter, J. H., J. Pharm. Sci., 60, 141 (1971). (66P) S iegel, H* E., Tonchen, A* E*, Clan. &em., 16,764 (1970). (67P) Spivak, V. A., Shcherbukhin, V. V., Orlov, V. M., Varshavskii, Y. M., Anal. Biochem., 39,271 (1971).
(6”) Stanley, P. E., ibid, p 441. (69p) Strmman, M., Ceci, L., TUCCi, A. F., Bwchem. J., 23,484 (1968). Stryer, L., Mol. Prop. Drug. Ciba Found. Symp., London, England, Jan. 27-29, 1970. ( 7 1 ~ Switzer, ) B, R., Summer, G. K., Clin. Chim. Acta, 32, 203 (1971). (72P) Taylor, K. LM., Anal. Biochem., 27, 359 (1969).
(73P) Thompson, J. H., Spezia, C A., Angulo, M., Ezperientia, 26,327 (1970). (74P) Waggoner, A. S., Stryer, L., Proc. Nut. Acad. Sci. U.S., 67, 579 (1970). ( 7 5 ~ Weil-Malherbe, ) H., ~ ~ ~ i~ ~ hchem. Anal., Suppl. Vol. 119 (1971). (76P) Yguerabide, J., Stryer, L., Proc. Nut. Acad. Sci. U.S., 68,1217 (1971). (77P) Yusemi, M., Delaney, W. E., Lindberg, M. A., Fashing, E. M., Anal. Chim. Acta, 44,403 (1969).
Chemical Microscopy George G. Cocks, Cornel1 University, Ithaca, N. Y. 14850
T
in this series (33) covered the two-year period from January 1968 through December 1969. This review covers the period from January 1970 through December 1971. However, there are included here a number of references which were omitted from the previous review. In this review an attempt has been made to include articles and books of potential interest to those who use the microscope to solve chemical problems. However, no attempt has been made to report publications in the fields of biochemistry, petrography, metallography, or ceramography unless they appeared to be of direct interest to chemists or chemical engineers. The chemical applications of electron microscopy are also omitted. References to publications in the fields of optics and crystallography are included if they appear to be of direct interest to chemists or chemical engineers. The great diversity and number of publications of interest to chemical microscopists make it nearly impossible to find and review all of them. Therefore we would appreciate comments or suggestions, particularly with regard to the omission of important published articles. We also appreciate the kindness of a number of persons who pointed out to us omissions from the 1968-69 review. During 1971 there was published, as an addendum to The Microscopist’s Newsletter, a bibliography of microscopy. This bibliography was extremely valuable to us in the preparation of this review. It is with extreme regret that we have to report that The Microscopist’s Newsletter will no longer be published. A new journal, Microstructures, published bimonthly by the A. 2. Publishing Corp., Los Angeles, Calif., appeared in 1970. In 1971 the Zeitschrift f iir Wissenshaftliche hfikroskopie HE PREVIOUS REVIEW
changed its name to Microscopica Acta, and in 1970 the Journal of Scienti$c Instruments became the Journal of Physics, Section E. BOOKS OF GENERAL INTEREST
A number of books which have in the past proved their value to chemical microscopists have been reprinted or revised and now appear as new editions. Among these are Johannsen’s “Manual of Petrographic Methods” (136) which has been reprinted. This is a reprint of the 1918 McGraw-Hill publication which has been invaluable to microscopists interested in crystallography. Another old favorite, “Dana’s Manual of Mineralogy” (127) has also appeared in its 18th edition. The new 4th edition of Hartshorne and Stuart’s “Crystals and the Polarizing Microscope” (109) contains new material on the Berek and Eringhaus compensators, on special methods for determining refractive index, and on the examination of hard compact materials. It also includes more information on hot and cold stages and on single axis rotation stages. The same authors have brought out a 3rd edition of their “Practical Optical Crystallography” (108). This book gives sufficiently detailed instructions so that it is good for potential microscopists who must teach themselves. Rath (222) has written, in German, a book dealing with the theoretical basis for optical crystallography in transmitted light. He deals with techniques which are well established and of proved usefulness to chemical microscopists. There are many good diagrams in the book. Clarke and Grainger (SI) have written a more general book entitled “Polarized Light and Optical Measurement”. Hallimond (103) has brought out the 3rd edition of his “The Polarizing Micro-
scope” which deals with the microscope as an instrument. A4nother old book, Fedorov’s “Symmetry of Crystals,” first published in 1890 has been issued by the American Crystallographic Association as a monograph (61). A new book of general interest to crystallographers is “Color and Symmetry” (178) written by Loeb. Buerger has brought out two new books; “Introduction to Crystal Geometry” (24) and “Contemporary Crystallography” (26). The first of these is suitable as a textbook a t the undergraduate level. Wooster and Breton (300) have published a 2nd edition “Experimental Crystal Physics.” Bollman (19) has written a book entitled “Crystal Defects and Crystalline Interfaces” and Henisch (115) has written on “Crystal Growth in Gels.” Three books dealing with the microscopical analysis of drugs have been published. Fulton’s book (76) is called “Modern Microcrystal Tests for Drugs: The Identification of Organic Compounds by Microcrystalloscopic Chemistry.’’ Stahl (266) has written, in German, a practical book dealing with the chromatographic and microscopical analysis of drugs, and Jackson et al. (129) have produced “Powdered Vegetable Drugs: An Atlas of Microscopy for Use in Identification and kuthentication of Some Plant Materials Employed as Medicinal Agents.” Another atlas concerned with inorganic materials rather then drugs has been written, in German, by Keune (160). It is entitled “A, Picture Atlas for Qualitative Inorganic Microanalysis.” Kapp (14.”) has produced a book entitled “How to Know Pollen and Spores” which contains 499 drawings of various specimens. Two books on photomicrography have been published. Loveland (180) has written a comprehensive treatise
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on photomicrography. This book is in two volumes and contains much information about microscopes and related equipment as well as material directly related to photomicrography. Gander (78) has published an English edition of a German language booklet first published in 1968. It is called “Photomicrographic Technique for Medical and Biological Students.” A compilation (21) of papers written by A. Bouwers contains several papers dealing with microscopy. FranGon has written two new books, one dealing with experiments in physical optics (71) and one, in French, dealing with holography (70). The latter contains information on holographic microscopy. A book “Laser Applications” (236) also contains a chapter written by Thompson in which holographic microscopy is discussed. Another book (14) entitled “Applications of Holography” has information which may be of interest to microscopists. The use of multiple beam interference microscopy for studying metals is the subject of a new book by Tolansky (878). A more general book on metallography entitled “Optical Microscopy of Metals” has been written by Gifkins (92). He covers reflected light illumination including interference and phase difference techniques, photomicrography, specimen preparation, alignment, quantitative microscopy, and the limitations of devices and techniques. The book is general rather than specific so it is not a manual for metallographers. Pepperhoff and Ettwig-Duisburg have published, in German, a book on interference layer microscopy (218). “Quantitative Stereology” is the subject of a book by Underwood (280). It deals with the interpretation of threedimensional structure from data obtained from plane cross-sections. Three books on the general subject of physical techniques in biological research seem useful to microscopists. Ruthmann’s book (837‘) “Methods in Cell Research” is a translation of a German book and includes microscopical methods. Martin and Welford (193) have written a section on the light microscope which appeared in “Physical Techniques in Biological Research,” 2nd edition, Vol. I, Part A. Part B is to contain a chapter by Osterberg and deVeer on phase and interference microscopy. A book by Slayter (269) entitled “Optical Methods in Biology” is intended to give biologists an understanding of the optical principles upon which microscopy, spectroscopy, and diffraction are based. It is a comprehensive book and is almost certain to be of value to microscopists whether or not they are interested in biology. 34R
ARTICLES OF GENERAL INTEREST
are discussed by Barrett and Yust
“Chemical Microscopy-A Neglected Tool in the Life Sciences” was the topic of a paper presented a t the 1970 meeting of the Optical Society of America by Carroll (87). McCrone discusses what a microscopist does and some of the tools he uses (186). He also discusses the “Choice of Analytical Tools” (188) in which he compares various analytical tools and their characteristics. Instructions on how to buy a compound microscope by Delly (48) includes a useful list of available objectives for transmitted light microscopy, fluorescence, phase and polarized light microscopy. I n connection with the analysis of feeds, Delly also discusses the use of the polarizing microscope, mounting media, light filters, dispersion staining, stereoscopy, top lighting, and chemical microscopy (4).He also describes a procedure for classifying particulate materials according to a six-digit classification scheme (46) Friedman (73) presents a general discussion of optical contrast methods in microscopy including polarized light, phase contrast, interference, and other methods such as dark field and Rheinberg illumination. In a .series of articles written in German, Appelt discusses the illuminating system of the microscope including its numerical aperture, dark field, illumination, and incident light illumination (9), condensers for bright field transmitted light (IO), condensers for transmitted darkfield, incident darkfield, and incident bright field illumination (11), light sources ( I $ ) , and light filters (18). Dahlke (39) describes, in German, a simple arrangement for measuring the effect of the field diaphragm. He concludes that up to N. A. = 0.50, the field diaphragm can be ignored. He also could not observe any influence of stray light from parts of the specimen outside the field. A similar series of articles has been written by Gerlach (also in German) on testing the mechanical parts and on the illuminating system of the microscope (87), the testing of objectives and oculars (88), the numerical aperture of microscope objectives (89) and how to measure the numerical aperture of objectives (90). White (892) has written on “The Photometry of the Microscope” treating light sources, methods of illumination, retinal illumination, illumination in the projected image, and the determination of exposure time for photomicrography. Young (301) considers the examination of surface topography using various tools such as transmission and scanning microscopes, interference microscopes, the Topografiner and contact stylus instruments. Some of the fundamental ideas in topology and their application to problems in metallography
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(16).
A symposium on the computer as an optical element in microscopy has been presented a t the 1971 meeting of the Optical Society of America ( N 6 ) . At the same meeting, there was a series of contributed papers on biomedical image processing. Papers on historical topics were presented by Schlueter (246) and Gause and Gorlich (81). I n Schlueter’s paper, there is an interesting tabulation of historical events affecting microscopy which occurred between 612 BC and 1800 AD. Gause and Gorlich cover Ernst Abbe’s relations with a few English microscopists of his time. Articles having some special interest to teachers of microscopy include one by Marden et al. (198) describing a sort of analog computer which calculates what happens in the image under given sets of conditions. The article is entitled “A Model Designed as a Teaching Aid for Phase and Amplitude Contrast Microscopy.” I n addition to articles mentioned above (87-90), Gerlach (91) has discussed image formation in the microscope. This article contains diagrams which should prove useful in teaching. Horrocks (121) discusses “drawing with optical aids” in a series entitled “Beginning Microscopy.” In this same series, White (894) discusses ;the microscopy of plant tissues. The series of papers by Appelt (9-18) and the paper by Dahlke (39) should also be of some interest to teachers. OPTICS
See Books of General Interest (31, 71, 869) and Articles of General Interest (S9,91,992) *
The effects of partial coherence of the illumination on the resolution of the microscope are discussed by Sirohi and Bhatnagar (267, 168). They conclude that the use of a light source having a certain nonuniform spatial intensity improves resolution. The dependence of resolution on obstruction ratio of annular apertures is presented graphically. Som (864, 266) has made a theoretical study of the influence of partially coherent light and aberrations on the resolution of the microscope. Mondal and Slansky (802) have presented, in French, a general solution for the calculation of the image formed of phase or amplitude edges observed using phase contrast with partially coherent light. Sakayanagi (839) has written a paper on the “Sharpness of an Edge in Microscopic Measurement.” Karovic (146) presents a paper, in German, entitled “Considerations on the Processing of Signals in Photoelectric Microscopes,” which deals particularly with the location of line marks. Osborn and Roberts (810) record some observa-
tions on the optical fringe effects a t prism borders in sections of tooth enamel, along with an explanation of these effects. Three articles on phase contrast imagery have appeared. De and Mondal (42) discuss “Phase Contrast Imagery of Circular Phase Objects in Partially Coherent Light.” Mondal and Slansky (203) have written a paper, in French, on the influence of the position of the phase ring in phase objectives on image contrast. Hartley (106) considers “The Place of Phase Contrast and Interference Microscopy in Image Formation.” Rienitz (232) has systematically investigated the manner in which image character in differential interference contrast microscopy depends on optical and instrumental factors. “Progress in Optics,” Vol. VI1 (897) contains sections on multiple beam interference and natural modes in open resonators and on image formation with partially coherent light. Wilcox and Izett (296) have written an article entitled “Optic Angle Determined Conoscopically on the Spindle Stage: I. Micrometer Ocular Method.” Rath and Pohl have written a series of articles reporting the mathematical reproduction of interference figures as follows; for optically active nonabsorbing crystals of any symmetry and orientation, in German (22S), for uniaxial crystals, in German, (227), for biaxial crystals, in English (f?%$), for biaxial crystals, in German (228), and for strongly absorbing crystals of low symmetry, i.e., orthorhombic, monoclinic, and triclinic, in German (225). A new technique for the analysis of the performance of a polarizing microscope with crossed Nicols is given .by Katti et al. (147). It involves the modulation of a periodic object of triangular wave profile and is a mathematical study. The same authors (148) present expressions for the intensity distribution in diffraction images of a general periodic rectangular wave object in a polarizing microscope with crossed poiarizers. The analysis of polarized light is the subject of three papers. Goldstein (95) discusses the analysis of polarized light with two quarter wave plates, giving apparatus and procedure. This method is highly precise and, although the author applies it to polarizing microscopy, it is of more general applicability. Pluta (217) reports on the accuracy of microinterferometric measurements of optical-path differences using a half shade. He obtains an accuracy of =J=0.003h or better using a shearing doubly-refracting interference microscope. Rath et al. (226) have investigated Berek compensators with calcite plates of correct and faulty orientation. A plate with faulty orientation gives
errors which are,particularly important when small phase differences are being measured. Three miscellaneous papers on optics were reviewed. Hewlett (117) discusses color and polarization systems for producing stereoscopic vision in standard light microscopes. Singh (266) presents an empirical relationship between the refractive index and the temperature of optical glasses. Ferguson (66) reports on a method of improving image contrast in metallographs. INSTRUMENTS
Microscopes.
A microscope used to study polymorphic transformations in high explosives is described by Faubion ( 5 9 ) . It consists essentially of a polarizing microscope with the light output monitored by means of a photocell. It also is arranged so that photomicrographs can be taken a t will. An “Ultrasonic Diffraction Microscope,” described by Rowe (236), is arranged so that the specimen is irradiated simultaneously with light and sound waves. With such an arrangement, particles having their refractive index near that of the mounting medium can be “seen.” Apparentiy the diffraction patterns set up in the specimen by the ultrasonic waves can be observed optically. King and Berry (151) describe a depth scanning microscope, and the nondestructive metrology of surface texture is described by Friedman ( 7 2 ) . “An Infrared Polarizing Microscope for Observation of Domains in Thick Samples of Magnetic Oxides” has been described by Enoch and Lambert ( 6 5 ) . Constructional details are given. With this microscope, ferrimagnetic structures in garnet sections up to 5 mm thick have been observed. Phelan and Dellen (213) have described a rapid scanning infrared microscope used for probing and mapping specimens, and Sherman and Black (254) have built a scanning infrared microscope using a He-Ne laser light source. This instrument presents the image on a television screen. Microscopes built to sense objects for various purposes are reported by Berg ( l 7 ) , Simpson (256), and Schedewie (243). The first author has built an instrument which automatically tracks and remains focused on an individual bacterium. It also records the motion of the bacterium along three orthogonal directions. The instrument described by the second author detects the displacement of an object in one dimension by photoelectrically determining the focal position. The third author’s instrument is an electro-optical high-speed measuring microscope using a laser light source. This microscope is used for highly precise detection of the edge of a line. It is used in integrated cir-
cuit artwork machines. The Nikon Co. also produces a device for automatically focusing lenses (20s). A vidicon microscope for counting fluorescent particles is described by Dyer and Fuller (64). The image is formed on a television tube. A new system for micro image conversion is described by Schlueter (247). It covers a wavelength range from 300 to 1200 nm using two image converter tubes. Dooley et al. (53) have combined two microscopes by attaching them to a common stage. Thus they have a comparator for fission track studies which can view the same location on the specimen and on the corresponding autoradiography a t the same time. Objectives and Oculars. McArthur (182) has designed a “Universal Measuring Eyepiece for the McArthur Microscope.” Essentially it provides for tube length adjustment so as to make the eyepiece scale correspond exactly to selected units. Gahm (77) describes a turret eyepiece to aid in the stereometric analysis of metal specimens. Delly (48) in an article on how to buy a compound microscope includes a list of available objectives for transmitted light, fluorescence, phase, and polarized light microscopy. Illuminators. Tither (275) in his article “Beginning Microscopy” writes in appreciation of the Lieberkuhn illuminator. Gray and Leslie (100) discuss the ‘Wetallographic Applications of Xenon Illuminating Systems,” recommending them for “older” metallographs. Stages. “A Large Granite Stage and Measuring Microscope” has been designed by Alston-Garnjost et al. (6). The range of travel is 0.5 X 0.5 m in the horizontal plane and 7.5 cm in the vertical direction. Accuracy is il pm in the horizontal plane and * 5 pm in the vertical direction. A leveling device for keeping specimens in focus a t 600X during automatic scanning is described by Jansen et al. (130). Jones (140) has designed a fiber rotating device for the examination of fibers either dry or immersed. A simple pressure chamber has been devised by Martin (194). It consists of a compressible ring between the slide and the coverglass. The cell is filled with water and compressed with a screw mechanism. Higher pressure optical systems are discussed by Van Valkenburg (282). Weibel (289) discusses sampling for stereology, then describes an automatic sampling stage. It will scan automatically or manually and in a continuous or stepped mode. Watts (288) also describes a motorized stage for microstereology. Julian and McCrone (143) discuss the factors, both instrumental and sample related, which can affect the accuracy of temperature measurements when
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using a hot stage. Woodard (199) describes the calibration of the Mettler FP2 hot stage. A number of hot stage designs have been reported; a hot stage for the study of gas-solid reactions (110), a thermoelectrically heated or cooled stage (61), and a warm stage (to 96 “C), in German (144). Three stages for rapid quenching studies have been reported (16, 113, 174). Jones (137) and Jones and Chadwick (138) have discussed gradient hot stage work suggesting new techniques for quantitative studies of solid-liquid systems. Delly (46) has suggested the use of aerosol cans containing liquified gases to cool quickly for chemical microscopical operations. Miscellaneous. See Arikles of General Interest (301). Burke et al. (26) have reported on “White-Light Three-Dimensional Microscopy Using Multiple-Image Storing and Decoding.” Kipping (164) reports, in German, on a “3D” condenser. Donato et al. (62) describes a “Television Display System for use with a Timbrel1 Double-Image Microscope.” Vieten (283) describes a projection system to be used in conjunction with the Zeiss TGZ-3 Particle Analyzer. This device eliminates the need for paper prints and speeds the analysis. An “Improved Piezoelectric Driver for Glass Microelectrodes” is reported by Rikmenspoel and Lindeman (233). It is used in micromanipulation of cells. POLARIZED LIGHT MICROSCOPY
See Books ( S l , 103, 108, 105, 127, 136, 222), Optics (53, 147, 1.48, 223, 228) and Instruments (65). Compensators and half shadow devices are described in an article entitled “Acessories for Measurement in Polarized Light” (173). Pluta (218) discusses, in German, the evaluation of birefringence and refractive indices of microcrystals by means of an interference-polarizing microscope with variable doubling of the image. An article by Hartshorne (10”) describes the principles of operation and the uses of the polarizing microscope including some crystdlography, some hot stage work, and some studies of aggregates. Smith (261) in part I of his series on “Color Contrast Methods in Microscopy and Photomicrography” discusses Rheinberg and polarized light microscopy for the study of biological as well as crystalline materials. ULTRAVIOLET AND INFRARED MICROSCOPY
See Instruments (113,,264). A “New Ultraviolet Spectrogram Microscope” has been designed and built by van den Bosch et al. (281). This instrument $onsists of monochromator (2000-7000 A range), as the light source, and reflecting optics which form an image of the specimen on the face of an image 36R
0
orthicon having a fused silica faceplate. An image is formed on a television tube, but in addition the transmitted intensity a t each wavelength setting is recorded. Mia0 and Longfellow (197) describe a technique which combines an Ar ion laser with an infrared microscope for the nondestructive testing of bonded connections on beam lead microcircuits. INTERFERENCE MICROSCOPY
See Books (212, 278) and Optics (106, 217, 232, 297). A general article on interferometry entitled “Measurement of Surface Relief” has been published by Leitz (170), and Steel (267) has written an article on “Interference Microscopy with Transmitted and Incident Light” in which various methods, including holography, are discussed. Tolansky (277) describes the preparation of thin anthracene crystals to be used as test objects for high magnification interference microscopy. They are useful for transmitted or reflected light. Pluta (215, 219) reports “A New Polarization Interference Microscope” which is a shearing type giving both uniform fields and fringed fields. The amount and direct’ion of the shear are continuously variable. Reflected-light differential-interference niicroscopy is the subject of a paper by Hoffman and Gross (120) in which they discuss the principles, use, and interpretation of images. Another article discussing incident-light interference microscopy describes an instrument which combines the ideas of Michelson and Linnik (171). This instrument is manufactured by E. Leitn, Inc. The Nomarski differential interference-contrast system, especially the instrument itself, has been described by Lang (169). A similar but’ more detailed description of the Zeiss-Nomarski differential interfersnce equipment for transmitted light has been written by Allen, David, and Nomarski (3’). In it they explain the functioning of the microscope and give directions for obtaining high quality images. In part I11 of his series of articles entitled “Color Contrast Methods in Microscopy and Photomicrography,” Smith (263) covers the Xomarski interference microscope, and in part I1 of the series, he covers “The Interference Microscope” (268). I n two articles Geissinger et al. (83, 84) describe a combination of Nomarski differential interference microscopy and fluorescence microscopy. I n the first article, they point out that this new combination is designed to improve resolution in fluorescent specimens. In the second article, they apply this tool to the study of cell culture monolayers.
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Piesch (114) reports that the Nomarski method of interference microscopy, using either brightness or color contrast, gave good results in the examination of etched nuclear tracks in various solids. Watkins and Tvarusko (187) describe a Lloyd-mirror laser interferometer which may be used to study diffusion in layers of materials. McLean (190) measured the birefringence of fibers using both a Leitz double beam interference microscope and a polarizing microscope with a compensator. The measured values did not agree. The author suggests a technique of measurement which seems to be satisfactory. HOLOGRAPHIC MICROSCOPY
See Books (14, 7 0 , 236). Cox (37, 38) has written two general articles on holographic microscopy. The first discusses three types of holographic microscopes in detail, the second, a review with 29 references, discusses types of microscopes, problems, and future possibilities. I n another paper, Cox et al. (36) reports on the use of cineholomicroscopy to study the microcirculation of blood in small animals. The arrangement permits frame rates up to 50 frames per second with a resolution of about 3 pm. An area image was reconstructed a t full size and observed with a microscope. Evans (58) has written a paper entitled “Quantitative Reconstruction and Superresolution of Red-Blood-Cell Image Holograms.J‘ Particle size measurement’s have been made using forward-scatter holography. They are reported by Stigliani et al. (271). Pavitt et al. (811) have described the holography 3f fastmoving cloud droplets. They used an in-line holographic setup with a &switched laser as a light source. The reconstructed image was viewed microscopically. Feleppa (62) reports on “Hoiographic, Motion-Induced-Contrast Images.” PHASE CONTRAST MICROSCOPY
See Optics (48,107, 205) and Articles of General Interest (132). A general article written, in German, by Hausler (112 ) has been entitled “Phase-Contrast, Microsccpy for Everyone?” In another article, also written in German, Woif (258) has described a simple device made from easily obtained parts which is capable of producing phasecontrast, images a t low power using a compound microscope. Fields up to 20 mm in diameter have been obtained. Two investigat’ors have combined phase-contrast with fluorescence microscopy. Nonce1 (201) describes, in French, a new apparatus for microcinematography. It is a two-beam instrument which permits cinemicrogiaphy in fluorescent light or in pha,se-
contrast, either alternately or simultaneously. Pluta (816) reports, in German, on combining phase-contrast and fluorescence microscopy. He gives n method of making the necessary apparatus and describes applications of the method. DISPERSION STAINING
“Expanded Uses and Applications of Dispersion Staining” are given by Goodman (96). I n a study of Aroclors he combined dispersion staining with hot stage use t o demonstrate how the identification of fusible compounds by the method of Kofler can be made more accurate. He also studied the effect of particle size on the sensitivity of dispersion staining. Forlini and McCrone (67) have applied dispersion staining to the study of textile fibers. The central screening method combined with polarized light was used. The data are tabulated and also presented in graphical form. ‘The identification of asbestos fibers by dispersion staining was investigated by Julian and McCrone (142). Material from a number of mines was examined. With more data, it may be possible to determine the source of asbestos fibers. FLUORESCENCE MICROSCOPY
See Instruments, Miscellaneous (54), Interference hlicroscopy (85: 84), and Phase Contrast hlicroscopy (216). In an article entitled “An Instrument for Quantitative Microspectrofluorometry,” Gurkin and Kallet (101) discuss the requirements for such an instrument and describe the Farrand Microscope Spectrum iinalyzer (?VISA). They also list possible uses of this instrument. Gander (80) discusses the importance of the correct choice of filters for immunofluorescence work. A theoretical approach to correction for light absorption within t,he object for quantitative microfluorometry has been presented by Ernst (56). Fuorescence standards are described by Sernetz and Thaer (252) and by Walker and Watts (885). The first authors describe a capillary standard and the second authors a fluorescent pigment mounted on a microscope slide. -4general article on fluorescence microscopy in biology and medicine has been written, in German, by Walter (286). MICROSPECTROPHOTOMETRY AND MICROPHOTOMETRY
See Ultraviolet and Infrared Microscopy (281) and Fluorescence Microscopy (101). Goldstein (94, 95) has written two articles on various aspect,s of scanning microdensitometry. The first article considers stray light (glare) and the second spot size, focus, and resolution. Weingiirtner (290) describes, in German, a laser microdensitometer, and Schlueter (245) de-
scribes the Koana-Nora approach to microspectrophotometry. The instrument uses a double beam and is manufactured by Olympus. Schiemer (244) reports, in German, on photometry with the Zeiss universal microspectrophotometer. A direct reading compact selenium cell reflectometer is described by Gavrilovic (82). At a wave length of 54 nm, the spot size is 30 pm. Harrison (106) describes an “Automatic Classifying and Histogram Plotting Equipment for Use on Microscopic Measurements of Reflectivity.” He used this instrument for analyzing coal samples. Faye (60) presents a “Semi-quantitative Microscope Technique for Measuring the Optical Absorption Spectra of Mineral and Other Powders.” Kuhnert-Brandstatter et al. (160) report, in German, on the microscopical characterization and identification of drugs by ultraviolet spectrophotometry. TECHNIQUES FOR SPECIMEN PREPARATION
Embedding and Mounting. The selection of specific embedding resins and cutting techniques for the study of coated papers is discussed by Quackenbush (220). The sect’ioning is done for light microscopical studies. Stockem and Komnick (272) relate their experiences with styrol-methacrylate embedding as a routine method for light and electron microscopy. Fengel (63) has investigated, using light microscopy, the changes that take place in water swollen cellulose fibers during dehydration and embedding. Mounting media are reported by several authors. Lindauer (177) considers polyvinylacetophenol ideal. Ores (209) uses Luft’s epoxy resin mixture, ordinarily used for embedding, as a mounting medium. Its refractive index is 1.488. Senior (250) uses polyester resin as a mounting medium. Whit,e ($93) has tabulated the refractive indices of various liquids and isotropic solids of interest to microscopists. Microtomy. See Embedding and Mounting (220, 272). Senicr (251) describes a method for reconstructing microscopic objects in t’hree dimensions by photographing successive serin1 sections on cine film using one frame for each section. When projected a t regular speeds, the viewer has the impression of passing through the specimen. Hills (119) ilses an ultramicrotone to prepare taper sections of metal samples. Such sections can be used to study the layers just beneath the surface of the metal. Grinding and Polishing. Cochran (52) describes methods of preparing plutonium for optical microscopy. Jedwab et al. (1%) suggest techniques for mounting and polishing small quantities of minerals. Lomas and Sim-
mens (179) use embedding followed by grinding as a means of preparing crosssections of textile materials. They describe the apparatus used to hold the specimen during grinding. TECHNIQUES FOR SPECIMEN EXAMINATION
Photomicrography. See Rooks (?8, 180), Articles of General Interest (292), Phase Contrast Microscopy (201), and
Microtomy (251). The “Characteristics and Choice of Photographic Materials for Photomicrography” is the tit,le of an excellent article by Loveland (181). Jenny (135) discusses, in German, the Schwarzchild factor for reversal color film. The origin of the reciprocity effect and the resultant change in sensitivity and color balance are explained. Average exposure factors and the nature of the color shift are reported for a number of 35 mm films. Smith (261263), in a series of articles, discusses “Color Contrast Methods in Microscopy and Photomicrography.” Booth (20) is concerned with photomicrography using a miniature camera. He discusses some of the problems encountered, especially with single lens reflex cameras, and suggests some solutions to these problems. Moellring (199, 200) describes the Zeiss Tessovar which is essentially a zoom system which can be fitted with various camera backs. There are also available a choice of light sources and stands. Kipping (155) has written, in German, an article about photomicrography with flash lamps. A number of articles on cinemicrography have appeared. Among them is a description by Dawsori et al. (41) 3f a modification of the shutter of a Bolex H-16 camera to make it more reliable. He also describes an electronic flashlamp and a method of firing it in synchrony with the shutter of the Rolex camera. Riddle (250) has written about “Int,errupted Lighting for TimeL,apse Cinemicrography.” He discusses the need for such lighting, the various means of obtaining it: and his solution t o the problem. Hara (104) describes a setup for fiiming both sides of an object a t the same time using time-lapse methods. Two cameras and two lighting systems are used alternately. Schwartz (249) has written a general article on time-lapse cinemicrography in biological research. A wooden base to hold the microscope, the lamp, and the camera for photomicrography has been designed and built by Kipping (152). Delly (47) tells how to use the field diaphragm to make stepped test exposures when there is no dark slide on the camera. Altmann ( 7 ) describes, in German, an easily constructed home made exposure meter for photomicrography, and Sauer (840) describes, also in German, a, flashlamp
ANALYTICAL CHEMISTRY, VOL. 44, NO, 5, APRIL 1972
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photometer for use in photomjcrography. Refractometry. See Optics (266), Polarized Light Microscopy (218), and Embedding and Mounting (293). Jones (139) has written an article on “Liquid Refractive Index Measurement Utilizing the Rotating Stage Scale.” With this apparatus, refractive index measurements good to about =t00.002can be made. Sunderman (273) discusses the determination of refractive indices of uniaxial crystals by orientation variation. Fusion Methods. See Books ( l o g ) , Instruments, Microscopes (69), Applications of Chemical Microscopy, Crystallography (28, 69, 102, 1.41, 161-163, 166, 166, 270) and Applications of Chemical Microscopy, Minerals and Ceramics (6, 206, 221). KuhnertBrandstatter, et al. (164) have investigated “The Effect of Protective Environments on the Determination of Melting Points of Decomposable Materials.” Fifty compounds were melted while protected by silicone oil, flowing nitrogen gas, or sealed in capillaries filled with nitrogen. The resulting melting points were compared with melting points determined in the ordinary way on a Kofler hot stage. I n many cases, the protected melting points differed from the ordinary ones. The use of silicone oil seems to be a good method for materials which do not dissolve in the oil. Kofler and Kolsek (166-168) present tables listing compounds and Kofler’s four physical constants for identification of the compounds. Also included in the article are some derivative reactions and some thermal data other than the constants given in the tables. Grabar (97) describes the crystallization of polyglycolic acid from the melt, and Grabar et al. (98) report on the thermal behavior of T N T as observed using a micro differential thermal analysis apparatus. McCrone (186) disagrees with Grabar regarding the thermal behavior of TNT. Particle Size. See Articles of General Interest (46), Instruments, Microscopes (64), Instruments, Miscellaneous (283) and Holographic Microscopy (211, 271). See also many of the articles reviewed in the next sections on Stereology and Automatic Image Analysis. Cheng and Sutton (80) discuss the absolute determination of particle size and shape. Schwartz and Elias (248) describe “An Optico-Electrical Particle Size Classifier” which consists of a diaphragm which is attached to a stepped contact. The diaphram viewed with a camera lucida is adjusted to fit a particle and on pressing a switch the size of the particle is recorded. Abrams (1) describes an improved, faster procedure for “Grain Size Measure ment by the Intercept Method.”
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ANALYTICAL CHEMISTRY, VOL.
Stereology. See Books (280), Instruments, Objectives and Oculars (77) and Instruments, Stages (288, 289). See also the following section on Automatic Image Analysis. Schaefer (242) presents the mathematical basis of stereology. White and Van Vlack (296) compare two- and three-dimensional size distributions in cellular materials. They combine computer calculations with experimental data and conclude that the pentagonal dodecahedron is the prototype shape of cells. Itoh (128) derives an analytical expression for the intercept length of cubes randomly dispersed in a matrix. Hennig and Elias (116) suggest a rapid method for the visual determination of the size distribution of spheres from the size distribution of their sections. This involves matching the histogram of the unknown particles with histograms of known size distributions. DeHoff and Bousquet (43) have developed procedures for the “Estimation of the Size Distribution of Triaxial Ellipsoidal Particles from the Distribution of Linear Intercepts.” These procedures have been developed for oriented, partially oriented, and random distributions. A method for the determination of axial ratios is outlined. Gander (79) gives more examples of micro-sterological investigations. Lang (167) describes the Zeiss Micro-Videomat; a television microscope for automatic stereometric analysis. He also describes how the instrument is used and what it will do. Automatic Image Analysis. See Articles of General Interest (276), Optics (146),and Microspectrophotometry and Microdensitometry (106). Andrews, Pratt, and Caspari (8) have written a book entitled ‘‘Computer Techniques in Image Processing” which covers optical data processing, digital optical processing, two-dimensional matched filtering, orthogonal transformations, image transforms, and image coding. General articles on image analyzing microscopes have been published by Stein (268)and Cole (36). A number of review articles on the subject of quantitative image analysis have also appeared; a general review by Jesse ( l 3 4 ) ,a review covering commercial metallic materials by Hougardg and Rose (126), a review covering nuclear materials by Jesse (136), and a state of the art paper covering medicine and biology by Mertz (196). Hillnhagen (118) has described, in German, how to conduct a lineal analysis in a completely automatic manner. “Instrument Errors in Quantitative Image Analysis” are considered by Cole (34). He discusses sources of errors, the manifestation of errors, the relative importance of the various errors, and the means available to minimize errors. Dinner and Kaye (60) 44, NO. 5,
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have written about “pattern recognition concepts for automatic image analysis in fine particle technology.” Numerous articles describing equip ment produced by various manufacturers have been published. Fisher (66) describes the Quantimet 720, and Evans (67) discusses the “Standardizsr tion of the Automated Quantimet.” Manalan and Glass (191) and Brown (29) describe the nMC system, what it can do, and give examples of applications. Leitz equipment is described in a number of articles (126, 172, 176, 176, 238). Lang (168) describes “A Universal Modular Electronic System for Quantitative Light Microscopy” which is manufactured by Zeiss. Applications of automatic image analysis are described by several authors. Smith (260) describes the use of the Quantimet to determine a single number to represent the dispersibility of a pigment in a plastic. A number of methods are presented. Foster and Evans (69) discuss the “Image Analysis of Clay Fabric by Quantimet.” Frohlke (74) has written on “The Determination of Characteristic Values for Manganese Sulfides in Free-Cutting Steels by Means of Quantitative Image Analysis.” Miscellaneous. See Articles of General Interest (121) and Optics (117). Improvement of the accuracy of vertical measurements under the microscope is the subject of a paper by White (291). He gives a new general formula for depth and thickness measurements and tells how errors can be minimized. Vertical measurements using the fine focus knob can be made to A2 pm. Adams (2) tells how a laser microscope can be used for ultramicrosampling. Material on the specimen is vaporized by the laser beam, collected on the coverglass, and analyzed by flame photometry, mass spectrometry, neutron activation, or a and y spectrometry. Zijlstra (302)describes a vibrating reed magnetometer which can be used for microscopic particles. “A Few Problems of Microhardness Measurement” is the title of a paper by Gahm (76). He examines the behavior of Wickers and Knoop microhardness testers with regard to anisotropy in the specimen. Hasson et al. (111) have studied the nucleation of crystals of compounds produced when fluorine reacts with copper a t various temperatures. Photomicrographs and cinemicrographs were taken to follow the nucleation and growth of the compounds. Davis (40) has used contact microradiography to study alloys. He uses a light microscope to examine the radiographs. APPLICATIONS OF CHEMICAL MICROSCOPY
Crystallography. See Books (19, 24, 26, 61, 108, 109, 116, 187, 136,
178, 222, 296, SOO), Optics (223-286, 227, 228), Instruments, Microscopes (69),Polarized Light Microscopy (lor), Specimen Examination Techniques, Refractometry (273) and Specimen Examination Techniques, Fusion (97, 98, 106). Brumberger (23) has observed what he believes to be evidence for rhythmic precipitation in spherulites of poly-1-alanine. Haleblian and McCrone (102)discuss the importance of polymorphism, particularly in the pharmacetutical industry. Shaffer (263) uses the microscope to observe and identify silicon carbide structures. He shows a relationship between birefringence and the relative fraction of HCP layers. Krc (169) presents the evidence for clathration in the solvate of pyruvinium pamoate. X-ray diffraction and infrared spectral data are also given. Cheng and Pigford (29) have investigated the “Purity of Crystals Grown from Binary Organic Melts.” Mixtures of stilbene and dibenzyl were studied microscopically using a thermal gradient stage as well as other techniques. Carter and his coworkers (28, 206) have studied the formation and migration of gas bubbles (voids) in KC1 crystals which were subjected to a temperature gradient. Several investigators have examined the polymorphism of various compounds as listed here: NHrNOa, 2,4,6,-trinitroresorcinol, pentaerythritol tetranitrate, pentaerythritol hexanitrate, pentaerythritol octanitrate and pentaerythritol decanitrate (69); mannitol (the optical properties of the three phases are given) (141);steroid hormones (solvation was also studied) (161163); sulfonamides and related compounds (the formation of mixed crystals was also examined) (166,166);triphenylphosphonium-3,3,4,4tetrafluor& 2,5-dioxycyclopentylide (270), Resins and Polymers. See Automatic Image Analysis (260). Reimschuessel (229)has written an article on “The Utilization of Scanning Electron Microscopy and Optical Microscopy in the Characterization of Polymers.” Uejo and Hoshino (279)report on the structure of biaxially oriented poly(propylene) film. Rider (231) has investigated the anisotropy in oriented poly(vinylch1oride). Textiles and Fibers. See Instruments, Stages ( I @ ) , Interference Microscopy (190), Dispersion Staining (67), Grinding and Polishing (l79),and Embedding and Mounting (63). Morosoff and Ingram (204) have measured the spiral angle of cottons and Hebert el al. (114)have studied “The Effect of Convolutions on Orientation Measurements in Cotton Fibers.” Rollins el al. (234) describe microscopical examination of abrasion phenomena in durablepress cotton fabrics. Both light and electron microscopy were used.
Wood and Paper. See Embedding and Mounting ’ (220). Hoster has written, in German, a series of articles on the microstructure of wood. Part I (122) covers investigational methods, Part I1 (123) covers the wood of coniferous trees, and Part I11 (lad) the wood of deciduous trees. Miniutti (198) has studied ultraviolet irradiated redwood surfaces using both light and scanning electron microscopy. Jarman and Canning (131) have made a cme study of the use of abaca (manila hemp) in paper making. There are problems with knots and clusters of fibers in the paper sheet when mechanically decorticated fiber is used to produce thin tissues. Minerals and Ceramics. See Books (127,136), Instruments, Microscopes (66), Microspectrophotometry and Microphotometry (60,82), Grinding and Polishing (132), and Automatic Imaging (69,136). The use of phase diagrams in electronics materials and glass technology is the subject of Vol. 3 of the series of books “Phase Diagrams-Materials Science and Technology” (6). Obst and Simon (2M) and Radczewski (,??%1) have investigated the behavior of feldspars and mixtures of feldspars with kaolin or mica, respectively, on heating. Obst et a2 (207) have studied refractories for oxygenprocess steel converters using physical micromethods. Used dolomite and magnetite refractories were examined by light and electron microscopy and by electron microprobe techniques. Photomicrographs of lunar rocks and minerals along with descriptions of the samples are presented by von Engelhardt (284). Foster and De (68) have carried out a light and electron microscopical examination of shear induced structures in soft and hard clays. Di Gallonardo and Vandervoort (49) report on the effect of a polar twin on the orientation of dislocation etch pits on a (1010) plane of beryllium oxide. Metals. See Books (92,278), Articles of General Interest (15),Optics (66),Instruments, Objectives and Oculars (77), Instruments, Stages (16, 100, 110, 118, l74), Microtomy (119), Grinding and Polishing (SI), Particle Size (l), Stereology (296),Automatic Image Analysis (74, 126) and Techniques for Specimen Examination, Miscellaneous (40,76, 111). McCall and Buchheit have made metallographic studies of artifacts from Ecuador (184) and from South Arabia (183). The Ecuadorian artifacts were Cu-Ag alloys while those from Arabia were Cu and bronzes. Allmand and Houseman (4) report a new method for identifying nonmetallic inclusions. After the metals are polished on diamond grits, they are cleaned ultrasonically and a thin film of an intermetallic compound or metal is deposited on the surface by
vacuum evaporation. The resulting interference colors enhance contrast. Forensic Microscopy. See Books (76,129, 146, 266) and Microspectrophotometry and Microphotometry (160). See also following sections on Air and Water Pollution and on Analytical Microscopy. Sutherland et al. (274)have written a review article on “Pharmaceuticals and Related Drugs.” I n this review, microcrystallographic methods for the analysis of drugs are cited. Air and Water Pollution. Ferguson and Sheridan (64) have written a booklet entitled “Optical Microscopy as Applied to Air Pollution Studies, a Reference List.” This list includes citations from books, pamphlets, and journal articles. McCrone (187)writes about “Microscopy and Pollution Analysis.” This article tells what a microscopist can do about identifying particulate matter and discovering its source. Biology and Medicine. See Books (193,237), Articles of General Interest (294, Holographic Microscopy (36, 58), Photomicrography (249), and Automatic Image Analysis (196). Miscellaneous. See Ultraviolet and Infrared Microscopy (197) and Interference Microscopy (214). Graft (99) has written an article on the “Microscopy of Integrated Circuits” in which the role of microscopy in determining the causes of circuit failures is discussed. Keen (149) reports on a light and electron microscopical study of the wear of diamond cutting tools used in cutting A1-Si alloys. He recommends a light microscopical examination during manufacture. ANALYTICAL MICROSCOPY
See Books (76,129, 146, 160, 266), Microspectrophotometry and Microphotometry (106, 160), Dispersion Staining (96, l4W), Techniques of Specimen Examination, Fusion (165168),Techniques of Specimen Examination, Miscellaneous (8, SO$), and Applications of Chemical Microscopy, Crystallography (69,97, 98, 141, 161-163, 165,166,186,263,270). By controlling the relative humidity in a small cell on the microscope stage, Chamot-type microchemical tests can be carried out in nanogram sized drops. This reduces sample size in such tests from the microgram to the picogram range. The techniques for carrying out these ultramicro chemical tests are described by McCrone (189). Mela and Sulonen (196) report “A New Method of Laser Microprobe Analysis.” Schaeffer (241) has used the reactions of 4,4‘-dipyridyldihydrochloride with the platinum metals and gold as a basis for microscopical tests for these metals. A solution of the reagent in normal HC1 will form characteristic crystals with
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low concentrations of these metals. Berisso (18) has investigated the formation of the complex FeIa.3CoH7N. Similar complexes are formed with Co(11) and Ni(I1). Using these complexes, the three metals can be detected in mixtures at concentrations of 1 to 0.01% in 1hour. Stevens (869)has surveyed the literature on organic compounds that have been characterized microscopically. The references are divided into two tables; one based on classification according to functional groups and the other based on authors’ names. Genest and his coworkers (85, 86) have reported microcrystallographic tests for alkaloids. Among the alkaloids studied are lupanine, isolupanine, hydroxylupanine, epihydroxylupanine, angustifoline, ormosanine, ormojanine, aad panamine. LITERATURE CITED
(1) Abrams, H., Metallography, 4, 58 (1971). (2) Adams, M. D., Microscope, 19, 157 (1971). ,----, (3)Allen, R. D., David, G. B., Nomarski, G., 2. Wiss.Mzkrosk., 69,193 (1969). (4) Allmand, T. R., Houseman, D. H., Microscope, 18,11 (!970). (5) Alper, Allen M.,Phase Diagrams; Materials Science and Technology, Vol. 3,“The use of phase diagrams in electronics, materials, and glass technology,” Academic Press, New York, N.Y., 1970. (6) Alston-Garnjost, M., Davis, J. W., Dauber, P. M., Smits, R. G., Rev. Sci. Znstrum., 43,1565 (1971). (7) Altmann, H., Mikrokosms, 59, 285 (1970). (8) Andrews, Harry C., Pratt, W. K., Caspari, K., om uter Techniques in Image Proces%g,R Academic Press, New York, N.Y., 1970. (9)Appelt, H., Mikrokosms, 59, 155 (1970). (10)Zbid., p 234. (11) Zbid.. D 318. (12)Zbid.; 60, 60 (1971). (13)Zbid., p 316. (14)Barrekette, E. S.,et al., “Applications of Holography,” Plenum Press, New York, N.Y., 1969. (15) Barrett, L. K., Yust, C. S., Metallography, 3,1 (1970). (16)Benscoter, A. O., el al., Microslructures, 1, 21 (1970). (17)Berg, H. C., Rev. Sci. Znstrum., 42, 868 (1971). (18)Berisso, B., Mikrochim. Acta, 5, 965 (1970). (19) Bollman, W., “Crystal Defects and Crystalline Interfaces,” Springer-Verlag, New York, N.Y. 1970. (20)Booth, A. D., Microscopy, 31, 298 (1970). (21) Bouwers, A., “Selected Papers,” North-Holland, Amsterdam, 1969. (22) Brown, J. F. C., Microscope, 19, 285 (1971). (23) Brumherger, H., Nature, 227, 490 11970). (24)Buerger, Martin J.,,,“Introduction To C stal Geometry, McGraw-Hill, New X r k , N.Y., 1970. (25) Buerger, Martin d., “Contemporary Crystallography,” McGraw-Hill, New York, N.Y., 1971. (26)Burke, J. F., et al., Nature, 231, 303 (1971).
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(27) Carroll, K., J. Opt. SOC.Amer., 60, 1553 11970). (28) Cader, ’R.E.,Rosolowski, J. H., Nadeau, J. S., J. Appl. Phys., 42, 2186 (1971). (29) Cheng, C. T., Pigford, R. L., Znd. Eng. Chem., Fundam., 10,220(1971). (30) Cheng, D. C., Sutton, H. M., Nature (Phys. Sei.), 232, 192 (1971). (31) Clarke, D., Grainger, J. F., “Pols;; ized Light and Optical Measurement Pergamon Press, New York, N.$,, *n“* 1YI1.
(32) Cochran, F. L., Microstructures, 1, 27 ( ). \1 -9 -7 .0 -,(33jcocks, G. G., ANAL. CHEM., 42, 114 (1970). (34) Cole, M., Microscope, 19,87 (1971). (35) Cole, M., AWT. Lab., June 1971, D 19. (38) Cox, M. E., Buckles, R. G., Whitlow D., Appl. Opt., 10,128 (1971). (37) box, M. E..Laser Focus, Feb. 1971, p 41. (38) Cox, M. E., Microscope, 19, 137 11971’1. (39)-Dailke, G., 2. Wiss. Mikrosk., 70, 80 (1970). (40)Davis, A. M., Metallography, 3,165 (1970). (41)Dawson, M., Johnstone, A. J., Matthews, J. E., J. Microsc., 91, 139 11970). (42)-D
