Review of Fundamental Developments in Analysis
Electron Microscopy T. G. Rochow, Ann M. Thomas, and M. C. Boffy American Cyanamid Co., Stamford, Conn.
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cuts across many scientific disciplines. Growth and activity are reflected by the increased membership in the Electron Microscope Society of America (49) and by the numerous papers appearing in the literature here and abroad. The enthusiasm about the 1960 meeting of EMSA a t Marquette University, Milwaukee, Wis. (August 29 to 31, 1960) and the more distant meeting of the International Congress for Electron Microscopists (August 29 to September 5, 1962), at which EMSA will be host at the University of Pennsylvania, forecasts continued active interest. EDUCATION
The acceptance of full semester college courses and opportunities for research substantiates and secures electron microscopy as a scientific domain. A summary (23) and a two-part article (14) have been published on a symposium sponsored by the American Chemical Society on “Microscopical Scientists: Their Education, Employment, Activities, and Microscopes.” Nomenclature, Units of Measurement, and Standardization. While a science is young and its language is being formed (8) there is extra freedom with responsibility to make changes whenever and as soon as desirable. Baker (19) wishes to change the basic unit of electron microscopical measurement from angstroms to millimicrons because the angstrom unit digit is believed insignificant in both practice and theory. While Wood appears t o refute (51) the insignificance, the millimicron does seem to be the logical extension of the metric system: kilometer, meter, millimeter, micron, and millimicron. Moreover, we do write and read in series of thousands, not tenthousands (19). Acceptance in America of the millimicron as the principal unit of electron microscopy could begin with EMSA (@) or ASTM Subcommittee 28 of E-1 on Microscopy (10) after some interested individuals initiate action. Testing resolution has long been a problem in electron microscopy. Recently Wood (61) used particles of platinum evaporated onto mica and
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transferred t o a carbon film. He demonstrated a resolution of 11.5 A. with the RCA EMU-2C fitted with Canalco (43) accessories and equipped with a highly stabilized supplementary power supply; the resolution was 8.5 A. operating the Siemens Elmiskop with the objective and intermediate lenses coupled and with Condenser I energized. Both instruments were corrected visually for astigmatism. Micrographs with the RCA microscope were taken a t 21,750X on Kodak fine-grain positive film and with the Siemens microscope on Gevaert contrast plates. Routinely, on biological specimens, Wood obtains a resolution of 20 A. or better and concludes that this compares favorably with the performance of the Siemens microscope. Standards of specimen (61), photographic emulsion ($), resolving power of a n electron microscope ( Z ) , and resolution in a n electron micrograph (3) were discussed and investigated further. Akashi, Masuda, and Tochigi (9) modified the method of Ruska and W O E (268) for testing resolution. Akashi et al. measured the distance between centers of many pairs of platinumpalladium particles and considered the distance resolved with 50% probability instead of the smallest distance resolved. With 50% probability half of the pairs of particles which are observed to be separate on a single micrograph are also perceptible on both micrographs. The reasons why some pairs of particles are not seen on both photographs are changes in specimen due to electron irradiation, defocusing during test, and granularity in photographic emulsion. The calibration of magnification is usually done by an engraved grating and the finest one generally available has a spacing of about 1 micron. For magnifications over 1O,OOOx, a smaller known unit is desirable. The superlattice manifested by a variety of antigorite (Yu Yen Stone) was described by Brindley, Comer, Uyeda, and Zussman (W), m-ho estimated the spacing to be 100 10 A. Uyeda, Masuda, Tochigi, Ito, and Yotsumoto (282) vacuumdeposited TlCl on the antigorite and obtained electron diffraction patterns of the two. Then using the spacing of TlCl as standard, they determined the
spacing of the superlattice of antigorite to be 89.7 A. By x-ray diffraction Espagne (54) calibrated the spacings of Menter’s (119) lattice-image of a view of p-copper phthalocyanine and reported 9.9 0.2 A. Suito and Uyeda (169) by electron M r a c t i o n report 9.63 A. for this view. Hashimoto and Yotsumoto (82) show that measurements in the center of the field are the most accurate. Labaw (62) refers to lattice-images as interference patterns, analogous to those obtained light-microscopically with a ruled grating. We believe that the importance of gratings to standardiaation, micrometry, and experimentation is also analogous in the two microscopies. Bassett and Menter (20) resolved the 020 spacings as 6.9 A. in molybdenum (VI) oxide crystals. To obtain a finer periodicity with metals they, together with Pashley @I), overlapped nickel and gold crystals and obtained moire patterns (163) with a spacing of 5.8 A. Measurement and standardization of vertical distances are also important. Ohh and Carroll (141) have recently measured the thickness of an anodic oxide film (Ni0.Cr20J on stainless steel of 0.05-mm. thickness by taking an electron micrograph and measuring the ratio of light intensity through a micrographic area representing a hole in the anodic film, to light intensity representing the film. They used equations based on the work of Hall and Inoue (72) and Lena (207) and report film thicknesses in the order of 6 to 30 A. Visibility. Resolving power of the microscope, nature and preparation of the specimen, image contrast, and resolution are all interrelated in final visibility. I n the past biennium there has been much discussion of electron microscopical visibility, exemplified in a symposium at the EMSA meeting in Santa Monica (60). Reisner (262) spoke of the physical basis and methods of improving instrumental contrast. Bahr (18) discussed contrast in their sections and R e u s (249) talked on contrast by shadow casting. Sixon (52) deduced from electron microscopical theory that it is possible to gain a factor of 10 in contrast while losing only a factor of 2 in resolution by operating a t 5 to 15kv. The theory was supported by
Wilska and Nixon (192) with experimental results (60, 91). The intensity of the image at 10 kv. was sufficient t o permit a &second exposure at 20,ooOX. This is important information regarding the best practical operation of the microscope. Final visibility, however, depends upon the nature and preparation of specimen (6, 58) and on electron micrography (3). C h a t t e j e e (58) evaluated many of the heavy metals commonly used in shadowing by relating their thickness, contrast, and grain size. Uranium was found t o be superior to chromium, palladium, gold, phtinumiridium, or gold-palladium. Zeitler and Bahr (198) found good agreement with the theory of electron scattering for carbon and elements of comparable atomic number, but not for elements of higher atomic number. Thus the application of the theory to biological objects can be considered workable with a n accuracy of =k1001,. Because the formulas of Zeitler and Bahr are in reasonable agreement with the measurements of Hall and Inoue (72), Valentine (185) deduced the conditions for minimum appreciable increase in contrast in the image of any part of the specimen. There are three bases on which to compare contrast : When selecting an element for shadowing with m i n i u m thickness, the element should be of great density. A heavy element is also best for use as a stain, provided that it is to be disseminated throughout a very thin specimen. For a relatively thick specimen, however, the percentage uptake of stain required for contrast throughout the specimen is almost independent of the nature of the stain. For a bacterium, for example, 2% of any stain would be worth trying, including the usual ones of light microscopy. The most useful stains are selective a t a membrane or interface and are not uniformly distributed as assumed. If this membrane runs through the thin section almost normal t o the section, then one atom of a heavy element such as osmium per 400 A.’ of thin section will be sufficient to increase contrast; hence, osmium is a stain in theory as well as in practice (185). In addition, Thornburg (&Stoeckinius I), (91), and Bahr and Zeitler (18) have studied the chemistry of osmium staining. Hall and Inoue (72) found that when the angular aperture of the electron microscopical objective was reduced from 4.2 X lo-* t o 2.1 X 10-8 radian, the scattering cross section of polystyrene latex increased by 3oy0. This is in good agreement with Lenz’s theory (107, 169). However, with a n element of high atomic number, the theory predicts that the size of the aperture will have little effect on contrast. Valentine (f8.4) not only confirmed this empirically, but he suggested a method
of ditrerentiating substances of high atomic number from biological matter or other material of low atomic number by taking two electron micrographs of the same field using identical photographic conditions, but a different size of aperture for each micrograph. If the specimen as a whole is more opaque than the supporting film and the photographic exposure is sufficient, an area of specimen showing no change in opacity with change in aperture may be identified with a n atomic number greater than 20. Color contrast can be achieved by projecting the two images by different colors of light--e.g., red and green-and superimposing the two projections. TECHNIQUES
Preparation of Specimens. Purposely fracturing specimens to reveal internal morphology and structure has been used for some time in ceramography (41) and resinography (166). Bierlein, Mastel, and Turkalo (60) continue to use fractography in metallography (197). Recently Anderson and Holland (61) have fractured undrawn fibers and bristles of Nylon 66 and have found that manifestations by fractunng from liquid nitrogen are essentially the same as by a combination of cutting, polishing, and ionic etching a t room temperatures. However, ionic etching revealed more spherulitic detail while fracturing revealed a peculiar platelike texture. I n brittle fracturing of fibers, however, the view may be determined by the fiber architecture rather than by the direction of applied force. Presumably by stressing crosswise, drawn embrittled nylon fibers broke longitudinally. Scott’s microdissection (146) of synthetic fibers is not strictly comparable, but it is interesting that he nicked fibers diagonally and found (at room temperatures) that they peeled longitudinally. Steere (61) has fractured frozen biological specimens as an alternate to or confirmation of microtomy. Krogh-hloe (100) and Bierlein and Mastel (61) found fractography useful for ceramics, glasses, and like materials. Stover and Halick (61) summarize that the path of fracture in a brittle material frequently reveals features of structure which are difficult to observe by other techniques. They caution that interpretation requires additional information and an understanding of the three classes of fracture : conchoidal, intergranular, and cleaved. Quartz is generally thought to manifest conchoidal fracture. The National Bureau of Standards cleaved quartz and published a micrograph so artistic that it was used to initiate the practice of pictorial cover8 for Science (16.2).
The techniques of microbiology and of industrial microscopy are frequently
interchangeable. A good example is the microtomy of metals using microtomes which were originally designed by biologists to cut tissues. FerdndezMor& (66) has cut ultrathin sections of germanium with a microtome equipped with diamond knife and has demonstrated that the sections are useful in electron microscopy and diffraction. Tsuchikura (177) has cut aluminum, copper, and steel with a glass knife. Ionic bombardment as a method of etching metals has been reviewed by Bierlein, Newkirk, and Mastel (16), who extended the method t o nonconducting materials such as refractories, ceramics, and reactor fuels (61) by simply placing the specimen under wire gauze in the apparatus (26). Krypton was used as ionizable gas at a very low pressure. Anderson and Holland (61) used argon or residual air in a commerdally available vacuum evaporator equipped for a glow discharge to etch some fibers and bristles mounted in brass electrodes. Etching results were compared with those of fractography. Bierlein and Mastel (61) also sought and received information or confirmstion by old-fashioned chemical etching with hydrogen peroxide. The quantitative electron microscopical techniques originating with Backus s have well withstood and W ~ m (17) the test of a decade. The basic method of quantitative light microscopy (116) was used, which employs as reference substance a suspension of a known number of particles per unit volume or weight. By mixing a known proportion of reference substance with the particulate specimen and counting the number of reference and specimen particles, the concentration of specimen can be calculated. Recently, several investigators have reevaluated the electron microscopical method of Backus and Williams. Nixon and Fisher (137) contend that in the original method nearly all of the droplet-traces are too large to be included entirely in a field a t the desirable magnification, and that catching enough droplets on the tiny screen is unhandy and uncertain. The reference particles of polystyrene latex are too large, esbecially if the droplets of samLle are reduced for the first reason. Serumstabilized droblets are sprayed from an artist’s air-brush and sorted by a cascade impactor ( I 1 6 ) . The accuracy of diameter of standard latex (DOW BOG) is still questioned (18) and unansnered, but the finer LSO40-\ latex (27) is recommended and the diameter is taken as 850 =t80 A. Williamson and Taylor (189) consider especially the statistical aspect and, recommend that the ratio of specimen particles to reference particles be nearly unity. They show that the accuracy does not suffer significantly by varying the ratio within VOL. 32, NO. 5, APRIL 1960
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one order of magnitude, and they discuss a test to detect inadequate mixing. For determining the size distribution of nonoolatile liquids immiscible with water, Harris (77) collects the droplets on a wet film of gelatin and washes them away after the gelatin has set. Replicas are taken of the impressions, and the replica-diameters are converted to d r o p letdiameters by application of a spread factor borrowed from light microscopy. Osmium fixatives of biological tissue were re-evaluated by Scott and Nylen (169)on the basis of a previous physiclogical observation: hlueller and SzentGyorgyi (128) had noted that muscle fibers which had been dehydrated in cold acetone for three weeks and in cold ethyl chloride for one week and dried in air had the unique property of contracting after rehydration. Using this procedure instead of the buffered osmium(VII1) oxide one, Scot't and Nylen obtained wider bands of collagen microfibrils manifested much closer together. The texture obtained without osmium more nearly resembled that observed by Pratt and Wyckoff (f47)on teased native collagen fibrils and by Gross (68) on reconstituted collagen. Cryofixation n-as developed by Fernhndez-MorAn (511 to improve on low-temperature preparation and preservation of biological specimens. Tissue was frozen rapidly in liquid helium(I1) at about -270' C. and stained in isopentaneat -150' C., the ice-matri.. was replaced with solutions of heavy metals in alcohols or acetone at about - 100' C., and the specimen was embedded in a resin at about -50' C. He claims that helium(I1) not only freezes a t 100' C. lower than liquid nitrogen plus isopentane coolant, but it is more fluid and more heat-conductive. Meryman (1.20) describes a method based on the principle of vapor pressure gradient for sublimation freeze drying without a vacuum. It has the advantages of mechanical simplicity, speed, low cost, and simultaneous treatment of several specimens. The use of Araldite epoxy resin having suitable hardness, solubility, and penetrating qualities as an embedding agent for ultrathin sections is described by Glauert and Glauert (65) and by Moore (50). Shadowing of the specimen or its replica continues to be populsr, not, only for increasing contrast in the image, but also for deducing shape and height of the specimen (SI). Preuss (14.9) carefully considers errors in the assumptions that: a symmetrical spherical dispersion of the eraporant exists; the line-offlight of evaporant is linear; the evapcrant comes from a stationary roint source, is pure, homogeneous, and structureless; the shadow contour is a n exact geometrical projection of specimen profile; and the optimum thickness of 94 R
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evaporation is on the specimen. T o the study and remedy of these effects h e w has contributed a great deal by the use of pinholes as in a camera. As the emanation he uses the vacuum-diffused metal from the shadowing source which goes through the pinhole diaphragms. For replication, Bradley has modified (29, 91) his method by evaporating platinum together with carbon. Calbick (51) has confirmed that there is an increase in resolution which is even greater if evaporation is through a masking aperture. Murray (129) and Fourie (60) relocate areas for replication. Better control over the behavior and quality of substrate materials represents a significant advance in techniques. Cirbeck (91) copolymerized styrenes with variable amounts of either vinylpyridine or styrene sulfonate to obtain strong, thin substrates containing varying numbers of charged groups. By this method substrates are made which are more hydrophilic and better wetted by certain specimens in aqueous suspensions. Rhodes (5f)prepared and used carbon lamp filaments to make carbon substrates t$hich are claimed to have better reproducibility than those made from the usual graphite rods. Renshaw and Schneidmuller (50) found that the rate of evaporation of carbon was the most significant factor in controlling the intrinsic density of carbon films. Micrography. Electron micrography still lags behind light micrography by the use of glass plate negatives instead of flexible film which has the adrantages of being nonbreakable, less bulky, lighter, thin, and printable from either face practically without parallax. There are some disadvantages to film: less dimensional stability, potential curl, less resistance to scratching, and less choice of commercial photosensitive emulsions. Wood (51) recommends polyester film over cellulose acetate film not only for better dimensional stability but for lower moisture content enabling more rapid degassing. Specifically, he recommends D u Pont Ortho A and Ortho B Litho emulsion on Cronar polyester base of either 0.004- or O.OOi-inch thickness. The 0.007-inch stock is rigid and handles almost as easily as glass. The D u Pont Ortho A and B types are useful for extremely lon contrast specimens. For ordinary specimens he mentions D u Pont or Kcdak fine grain positive emulsions but, t o date, they were not available on polyester a m , Ilford Info recommends Ilford's fine grain safety positive film but does not state the nature of the film base. I t also gives recipes and directions for development. Nartin and Allred discuss darkroom techniques (114) and evaluation of negatives, choice of positive material,
and exposure time in printing by the aid of two standard charts (51). Hall (13) critically tested his enlarger, made improvements on it, and devised specific unsharp masks to control halation from negatives which were too contrastable and, for some reason, could not be taken over with less contrast. -4simportant as photomicrography is to electron microscopy there must be a perspective of which comes fist. These reviewers have the impression that there are still investigators who do their microscopy by merely examining micrographs, making micrographic studies, or preparing samples which produced a number of pictures. Photomicrographs like micrographic drawings can, a t best, illustrate under certain specific conditions \That can or should be seen under a sufficient variety of conditions and behavior. At lower requisites of vhibility, a t least, it should be with more confidence and less time and expense to take only a few typical micrographs t o illustrate what one sees over and over again on the fluorescent screen. ELECTRON-LENS MICROSCOPES AND ACCESSORIES
Instrument makers continue to accent versatility in the design and function of their electron microscopes. A fivecomponent electron diffraction adaptor described by Reisner (151) has been made available by RCA for users of the RCA EMGl and EhIU-3 instruments who require additional refinements in the electron diffraction patterns produced by crystalline materials. The versatile stage has four degrees of freedom and can be used for both transmission and reflection electron diffraction. The adaptor incorporates a charge neutralizer and a heater for increasing the specimen temperatures to 900" C. in addition to other useful features. After permanent installation of the power supply and control panel, all that is required is the slight modification of certain lens systems and the insertion of the diffraction chamber in the place normally occupied by the intermediate lens. A special aperture is then positioned in the standard specimen holder, thus converting the electron microscope into a dual purpose instrument. If alignment procedures are not too critical, the RCA adaptor should prove to be a worthwhile accessory.
Gulbransen and Copan (51) have used the RCA electron diffraction adaptor for the study of several metal oxides, particularly oxides of iron and chrcmium, and have reported favorable results. Using 100-kv. electrons they claim good instrumental precision, loKer background of inelastically scattered electrons, smaller electrostatic charging effects, and smaller diffraction angles for reflection studies.
Lodge and Havlik (110) modified their Model EXIC-BA console electron microscope, so t h a t they could obtain electron diffraction patterns from specimens placed in the conventional specimen holder. By removing the entire 1:ole piece, inserting a platinum aperture, and making a simple electronic modification, they obtained a diffraction pattern of a polycrystalline sample of magnesium oxide. Csers of this console instrument now have a n opportunity to establish the presence and Po&bly to identify finely divided crystalline materials in a sample a t very little cost and effort. Philips Electronics significantly extend the range and application of their E1I-75B electron microscope by enabling it' to be conT-erted to a projection microradiographic microscope ( P l I R ) ha;ing a resolution of 1 micron or better over a range of 2 t o 20 kv. Tn-o users of the Philips' EM100B h w cleverly modified the specimen hol' ler t o provide specific posit'ioning of the s1;ecirnens. For electron diffraction. Kalter (18511 altered the holder that crystalline specimens could be oriented t,o h a r e two degrees of rotational freedom. Wilsdorf (290) modified the holder so that it. functioned as a straining device permitting the direct observation of their metal foils undergoing plastic deformation. Significant I:hsiiges-i.e., indication of strain within the crystals in a n aluminum foil-support the potential usefulness of the method to those who investigate the behavior of metal or other plast.ic materials. To many microscopists t h e Siemens Elniiskop I represents the optimum in electron microscopes. Several recent improvements and additional accessories should make for greater versatility a n d convenience of operation. These include a rapid stage drive for scanning the specimen and for a greater traverse of the stage, a safety device for the vnciiuni system, a n additional controller for the intermediate lens, a meter for showing exact magnification, a universal diffraction unit with a stage that can be !ieated t o 1200" C., and an ohjectcooling device t h a t reduces the temFerature in the vicinity of the specimen to :I$ low as -80' C. This last-mentioned tlel-ice reduces the vapor pressure of iiyi.lrocarbons near the specimen, there11). minimizing cont,amination. -11tliough use of the cooling device requires 111ili'e time, its application is practically a i . ~ q i i i ~ i for t e the attainment of optimum resolution of detail in selected specimens. .i feature of the JEM-SY (Japan Electron Optics Laboratory Co., Ltd.) is that i t can be converted to a reflection niicroscope, thereby permitting the direct examination of surfaces. This a t t x h n i e n t consists of two sets of
deflection coils nhich are fitted to the upper part of the specimen chamber. Without apparently changing the tilt of the gun, the path of electrons can be changed so that it can be projected a t a n y angle less than 30" onto a specimen in a modified holder. The relatively large angle t h a t the electron beam makes with the axis of the lenses minimizes distortions. Ease of operation and a resolution of 500 A. are claimed. The utility of the method was demonstrated by micrographs of a copper alloy and pearlitic steel. If installation and operation are not too cumbersome, t h e electron reflection method should alleviate much of the tedium involved in making replicas of surfaces of metals. ceramics, fibers, and treated pulps. Haine, Agar, and hiulvey (YO) have designed a compact beam shift and tilt system. using electric and magnetic fields, to permit reflection microqcopy up to angles of beam tilt of 20". .% though their deflector system was applied t o an e'xperimental instrument, they provide considerable information and show results which should encourage other microscopists who might be contemplating modifications of their commercial microscopes. Further details leading to the correction of aftigmatism in magnetically deflected electron beams are described by Archard and Mulvey (16), who have used circular pole pieces from which semicircular portions have been removed. An x-ray microscope unit is available as a n accessory for the Hitachi, Type HU-11 electron microscope. According to the ray diagram, the intermediate lens focuses a small spot of electrons on a metal, reflection-type target, inclined at a 45" angle t o the alds of the column. The electrons generate x-rays which are preferentially attenuated by the specimen and are finally recorded on a photographic film. Other details are given by hlorito and Komoda who used a Type HU-10 instrument (186) a t varying excitation energies and with different metal targets to obtain r a d i e graphs leaving a resolution of several microns or better. They show several worthy illustrations of their results. The Transcope TRS-50 El has coaxial alignment of all lenses and aperture which is claimed t o be permanent. All lenses are fully corrected for aberrations due to anisotropy of magnetic materials and two specimens can be inserted simultaneously into the specimen holder. 'Axile these and other features should provide for ease of operation, the degree of permanence of alignment and lens stability when subjected t o routine cleaning may still have to be ascertained. For experiments requiring temperatures u p t o 3000" C., the heating and gas treating device of Hashimoto, Tannka, and Yoda (80) should be of interest. I t o and Hiziya (92) have modified a
commercially available heatable stage so that light gases, such as air or hydrogen, may be reacted with the specimen while it is being studied in tlie electron microscope. Perhaps this modification may make i t possible to examine a specimen while it is moist or drying (105). If so, a great many more kinds of processes may be directly observable. Before 1958, Russian-made electron microscopes, the ELI-3 and the EM-100, had no compensators for correcting lens astigmatism, preventing a resolution of better than about 50 A. (112). Improvements nere exhibited at the U.S.S.R. Exposition in Sen- York in 1959 ( I & ) , in the EM-5 with a maximum accelerating voltage u p to 60 kv. and rated resolution of 20 *4.,and the UEMB-100 (kv.) with a rated resolution of 15 A. The technique of autoradiography has been well established. However, the recent work of O'Brien and George (140) is of special interest as it places a certain dependence on the electron microscope. They modified the earlier m r k of Liquier-Lfilward (109) by applying a liquid photographic emulsion directly to the surface of ultrat'hin sections of radioactive yeast ceils already mounted on a specimen grid. After exposure for several weeks to rahoactive polonium210 in the section, the dry eniulsion film still in contact with the specimen was photograpically developed and then examined in the electron microscope. Their results suggested that y e s t cells were essentially impermeable to the radioactive material, because the high density spots were generally confined to the periphery of the yeast cells. This combined technique should be of considerable iniportance Lo those who are seeking microtracer methods. Electron Interferometer. Cosslett (4.9) describes the principle and shows the results obtained with hIollenstedt's (123) electron interferometer, which is being converted into a n electron interference microscope. According t o Cosslett the interferometer is analogous t o the Fresnel biprism. A narrow beam of electrons is split by a potential distribution of - 10 volts applied to a met,allized quartz fiber. The split, coherent b e a m then converge and interfere. Fringes are produced whose spacings depend on the applied potential and on t h e beam voltage. K h e n a very thin film of metal or carbon is placed in the path.of only one beam, a fringe shift is produced which is related to the electron optical path difference. Interference images were obtained by Mellenstedt and Biihl (123) which depict a phase shift in the electron fringes at the edge of a carbon film resting on another film. The methbd needs refinements, b u t it offers t h e prospect of investigating the coherence conditions in the electron beam, the local variations VOL. 32, NO. 5, APRIL 1960
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in thickness, and the inner potential of the specimen. APPLICATIONS
Microchemical Analyses. Chemical electron microscopy should begin where chemical light microscopy (113) leaves off. In practice this is n i t h particles of micron sizes. Of four analytical methods, electron diffraction is highly developed, while chemical, electron optical, and x-ray micromethods are still being developed. The four are complementary rather than competitive. The chemical methods are still limited to information about ions, but they are inexpensive techniques aud well adapted to the identification of discrete particles such as in air and industrial effluents. Lodge and Taft (111) have recently reviewed these complex problems of particle size, chemical composition surface properties, and their relationships. Vnlike chemical light microscopy n hich leans on characteristic crystalline habit and optical properties, chenlical electron microscopy depends chiefly on microspot tests made on a suitable mbstrate. Generally, the particles are reacted with acid and reagent t o form a characteristic spot or ring. If the spot or ring is not visible and recognized under the light microscope, an electron microscope is employed. Tufts and Lodge (180) showed that a chloride ion could be detected by an electron nlicroscope in sodium chloride in particles as small as 50 A. on a side which approaches the minimum for stable sodium chloride particles in air of maritime character. Sulfate particles down to loo0 A. and typical of industrial or volcanic origin were identified. The size of any resulting spot is approximately a linear function of the size of the original particle (predominantly of elements of the 6rst three periods of the periodic table). Hence particle-size distribution as well as particle number may be determined for each species. By the Same general method, Tufts was able to use the light microscope to recognize and measure particles resulting from lead-bearing (178) particles in city air and micronsized particles bearing krypton (179); her results are nevertheless of interest to electron microscopists. I n connection with the use of silver iodide to control rainfall, Koenig (98) developed a n electron microscopical method which is sensitive to 3 X lo+ gram of silver iodide. He reduced the silver halide by means of photographic reagents; the resulting twisted fibrils of silver are of characteristic habit. Frouls, Bowler, Bush (60), and Boraaky (60, 61) have all been very much concerned with the collection and study of air-borne particles, with relation to origin and nature of atmospheric pollution. 96 R
ANALYTICAL CHEMSTRY
Takiyama (I?’.$) is studying the change in habits of gion-th and dissolution of precipitates n i t h respect to variations in t h e and physical-chemical conditions. In a series of 12 articles he has described studies with such small, scarcely soluble crystals as vanadium pentoside, gold, and barium sulfate. At first he was repeating classical experiments and demonstrating wellknown principles such as the similarities between habits of growth and of dissolution, especially a t corners and edges rather than faces. Now he has demonstrated, n ith barium sulfate at least, that dissolution as well as growth starts and develops on the inside rather than the outside of a crystal. K i t h a colloidal sol of vanadium pentoxide as the example, he concluded that growth from small particles t o larger ones occurs by recrystallization rather than by coagulation. By electron diffraction he Froved that the transformation is not a change in phase, so it must be merely a change in habit. The particles in the gold sol were monodispersed. After nucleation the number of particles remained constant and their growth resembled autocatalysis a t their surfaces. Such fundamental results should be of value in many chemical technologies such as catalysis and polymerization. Suito (166) has summarized this and other work in identifying chemical composition and crystalline phase by correlation of electron microscopical morphology and electron diffraction. The study of particles a t or near the limits of Yisibility of light microscopy has progressed over the past two decades from the mere examination of, say, a pigment mounted in a sometimes arbitrary liquid to such applicable studies as the effect of pigment size and concentration on the weathering of automotive enamel by Teague (51). Haber (69) centrifuged diesel fuels and estimated the size and amounts of sludge. A great many methods for measuring and classifying particles according to sizes have been calibrated with light and electron primary standards in a published M T h f symposium (1.2). Aluminum hydroxides were prepared by RIoscou and van der Vllies (1.27) in five different ways and were aged in boiling water or heated in dilute ammonia. The products were found to be either in a spherical or fibrous amorphous habit or in a network of thin sheets, the Boehmite phase of AlO(OH), depending upon the method ilsed. Renshaw (60) found unusual pore textures in anodized aluminum oxide. The growth of these rosettes of pores was correlated to the anodizing current drain and to the properties of the aluminum oxideair interface. An analogous study by Cook, Nye, and Leonhard (50) shows the morphology of nickel powders produced by thermal decomposition
of nickel carbonyl, Ni(CO),, by electrolysis, or by chemical precipitation. They deve1oI;ed a replicating technique to show the three-dimensional dendritic structure characteristic of the carbonyl process and correlated their findings to the properties of sintered nickel powder plates for storage batteries. Hamilton, Brady, and Hamm (74,76) have published their fundamental studies of the latent photographic image. Chemical etching n-ith potassium bronlide, sodium sulfate, potassium cyanide, or sodium thiosulfate was enhanced by strain but there was no evidence that normal grains are part polycrystalline or highly imperfect, as other investigators suggested. Experiments on pitting by print-out exposure indicate that iodide ions provide internal recombination centers (76). Lawless (51) studied the chemical decomposition of thin platelet crystals of hloOl to RfoOz when heated in a vacuum. Platelets of hIoOz are formed along specific crystallographic directions of the MoOa, which is subsequently consumed by recrystallization to LIoOz. Identification of the oxides was accomplished by electron diffraction; moire patterns were frequent’ly observed. hIoir6 patterns continue (153) to occupy the attention of investigators of crystals, such as those of copper(I1) and zinc(I1) sulfides. Hashimoto (78) demonstrated with two light-diffracting gratings that the zigzag pattern can be made n i t h a defect in spacing of one grating twisted slightly over the other. He thought that moire fringes obtained electron microscopically are due to dynamic interaction of electrons with the surface of the crystalline specimen. A little later Hashimoto and Naiki (79) demonstrated that “ghost” dislocations could be obtained by covering a single grating with wrinkled transparent paper and photographing the combination. Increased effects were obtained in outof-focus images. They feel that previous workers (163) have electron m i c r e graphed true dislocations in crystals, but that the possibility of “ghost” dislocations should always be eliminated by a series of through-focus micrographs. Electron Optical Properties and Crystallography. Menter (119) observed electron microscopical images of periodic structure in crystals of metal phthalocyanines. Suito, Uyeda, Katanabe, and Komoda (170)have reviewed Rlenter’s interpretations and applications, as well as their own (168), and to them the images represent the crystal lattice of large molecules. They have found a contiguous variation in spacing of about 9.4 A. corresponding to the 20T face, and variation of about 12.6 A. corresponding to the 001 face. They observed this sharp change h spacing in a different habit from the one used by Jfenter. He used crystals lying on the
largest face (OOl), but their crystals lay on a different face (maybe 201). Suito and L-yeda (169) conclude by bright-field and dark-field electron microscopy and by selected area electron diffraction that the boundary between spacings is a twinning plane in a new habit (obtained from carbon tetrachloride) of the beta phase of copper phthalocyanine. Their interpretations are based on earlier infornution, including their cinematic (181) electron diffraction and microscopy of the transformation from the alpha phase (by precipitation in water from a solution in concentrated sulfuric acid) to the beta phase (from pyridine). The lattice images such as are manifested by phthalocyanines (54,119,169, 170, 181), “Yu Ten Stone” (33, 182), and “poly-L-proline 11” (34) are interference patterns (Fourier images) as explained by Labaw (51) and Cowley and Jloodie (45). Burge (34) also brings in the role of the double refraction of such cr>.stals. Thus electron optical and crystallographic properties are contributing to chemical electron microscopy. There is a great deal of fundamental and t,echnical interest in the morphology and structure of graphite as “whiskers.” 1Ieyer (51) has shown electron microscopically and diffractionwise that whiskers consist of laminar single crystals. Bacon (51)grows such whiskers in a direct current arc in about 92 atm. of argon gas at 3860’ K. These have diameters from 0.1 to over 5 microns and lengths up t o 3 cm. Their tensile strengths are u p t o 3,000,000 p.s.i. Some appear to be tubes; others are ribbons. They may give moire patterns n-ith a spacing of 400 -4.which hleyer ( 5 1 ) explains b y interference betn-een undeviated and doubly diffracted elec’tron beams by two laminar crystals with a small rotational disorientation. Grenall (50) has observed dislocations in graphite and has made motion pictures to illustrate the behavior of fundamental interest t o crystallographers and metallographers. Sewkirk (5G) studied dislocations by x-ray diffraction microscopy. His esaniples n-ere ferrosilicon, pure silicon, and lithium fluoride. The latter crystalline compound is being studied for dislocations and defects by Gilman and Johnston (64)and a number of other investigators because i t can be etched defectively by neutron bombardment. Mineral and Ceramic Materials. Comer (40) and Bates (22) illustrate abundantly how morphology and structure are correlated to chemical-physical composition and treatment in their mineralogical studies. Bates studied 64 silicate minerals with a similar layer structure by varying chemical analyses. H e and Comer are very careful t o define structure as the arrangement of the
atoms or ions in a crystalline material. Comer illustrates with clay composed of kaolinite and halloysite that identification by habit is surer when the examination includes fresh fracture surfaces before there can be any segregation or pictorial loss due to further preparation. B y such fractography and replication he showed that a specimen of seventine had both tubular and particulate units and that this physical composition may be related to the bulk density. Bates and Comer (25) classify members of the serFentine group of minerals according to whether they are of fibrous or platy electron microscope habit. Maser, Rice, and Klug (51)have now confirmed the question opened by Corner, that chrysotile fibers are also hollow with an inside diameter of 100 to 200 -4. They embedded the fibers in hraldite polymer and cut ultrathin sections nith a diamond knife. The application of electron niicroscopy and diffraction to mineral engineering by Lawver and Samsel (105) is not only a n estension of the field, but introduces techniques such as using methanol for washing and etching watersoluble sylvite (potassium chloride), in connection n-ith concentrating the potash mineral from its ore for fertilizers. They also eyplain the interaction of surface-active agents such as caprylic acid on slime-contaminated sylvite by shoning that the agent preferentially covers the contaminant and thereby improves froth flotation. Ross and Christ (157) review the geometry required for the interpretation of electron diffraction patterns compared n-ith x-ray patterns. They nere especially concerned n ith single thin crystals as they exist in some minerals and rocks and carefully studied potassium chlorate and colemanite [CaB304 (OH)3 H 2 0 ] . Along the same lines Suito and Tlyeda (167) have been studying clay minerals by electron microscopy and diffraction. Comer, Koenig, and Lyons ($1) and Xemetschek (133) studied dynamically the changes in electron microscopic morphology and diffractive structure as kaolinite is transformed t o mullite, cristobalite (51), and other phases. Schwarts (51) reported on studies of the composition, morphology, and thermal treatment of B e 0 plus A 1 2 0 ~and Bierlein and RIastel (51) did a related study of GO2. Tennery and Anderson (175) examined the free surface (air interface) and the polished-etched specimens of ceramic barium titanate t o study the growth patterns and domains. DeVries a n d Burke (48) studied ceramographically polycrystalline BaTiO, as fractured a n d as polished and etched surfaces. They showed a banding which they relate t o stress during the transformation from cubic t o tetragonal phase. These
studies depend upon and go beyond others (36,77). Skatulla, Vogel, and Kegsel (164) studied glasses of NazO and B 2 0 3 and claimed t h a t the melt consists of tn.0 immiscible liquids in the region of 6 to 24 mole % ’ ?;azo. If any such phase separates, Krogh-31% (1 00) thought that the phenomenon should be more pronounced in the LizO and U203system, which is also more stable in air. He studied replicas of fracture surfaces of several compositions and found that the thermal history had a strong influence on the texture. The first s t e p of devitrification were visible before the glass became turbid to the unaided eye. When the system contained < 10y0 Li20 two liquid phases esisted in a narrow temperature range. This is a good esample of a dynamic rather than a static study and leads directly to the construction of a n illustrated phase diagranl. Metals. I n his summary of t h e proceedings of t h e Symposium on Internal Stresses and Fatigue in Metals a t t h e General RIotors Research Center, September 1958, Freudenthal (62) emphasized the importance of studying the interaction betn-een residual stresses and fatigue. Today there is a renewed sense of structure distinct from the classical approach based on the mechanics of the solid isotropic continuum. He illustrates with the work of Coleman (5G), Muller and Hirsch (83). They and their contemporaries are depicting the residual stresses which are primarily the result of mechanical response to external force. Freudenthal believes that such observations and measurements have more qualitative significance (proof of esistence) than quantitative determination of their intensity or change in intensity. The quantitative determination of intensity of residual stress is advancing; analytical evaluation and prediction of mechanical properties will follow.. Bassett, RIenter, and Pashley (21, 143, .lZ?),by use of moire patterns from overlapping thin, face-centered cube crystals of nickel and gold, have resolved periodicity as small as 5.8 A. These pioneers predict t h a t in this indirect way, crystallographic dimensions of even less than 1 -4.may be resolved. They discuss the limitations and potentialities of this kind of study while the specimen is being treated in the electron microscope. A decade ago Heidenreich prepared thin films of aluminum and its alloys and examined the induced strain and thermal changes. About seven years later Hirsch, Horne, and Whelan matched thermal changes produced on the specimen while it was being exposed t o the electron beam of the microscope. Nom Berghesan and Fourdeux (24) and others have advanced to controlling and measuring the heat produced on
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the specimen by a n independent electric heat,er. I n the meantime \Tilsdorf (191),hleakin, Sun, and Varker (51) have xatehed the deformation in many kinds of metallic thin foils while they applied mechanical stress with their modified specimen holder. They make their specimens thin electrolytically. This ancI;’or chemical t,reatment is the method used by Saulnier and Croutzeillcs (160) on commercial titanium, 1Ioe (51, 9) on magnesium alloys, noswell and Smith (60) on iron, stainless steel, a-brass, and aluminum, Ohh and Carroll (141) on stainless steels, and Reinier (150) on nickrl-iron alloys. Sicholson. Thomas, and S u t t i n g (51, 135) reduce foils of aluminum alloys electrolytically until holes appear and tlwn che~iiicallyclean away the oxides. Other investigators obtnin their films by w.aporation of the metal. This group inrlndes Pic.gcl (GO) working mainly with copLer. Phillips (51 with silver. Huber. Smith, and Goodenough (89) with niugnt-tic nllriy! I,intl~,ganrcl-.\ndersen (ZOcS) {vitii zinc. :i1iiI its p r d t l ( , t s of o.sid:itioii, and T:iknhashi and Ilihnmn ( 1 7.3) n-it!i various :iluminum-copper alliiys undcrgoing ~ ~ l i n transformations. se h o t h e r shape of n i t h l o r nict:illoid for study is the tliscretc, Ixirtic-le produced by dccomposition froin its chemirnl compound in the vapor phase. Lefker, Bills, :and Syv 106) i?rotlucetl siliclon particles in tlit’ir research on aeniiconductors. \\-at?(in and his associates pxaniined magnttic (787). colloidal, possibly radioactive particles of a-iron in cancer revarch (1,W). Kronenberg ancl Tenzcr i I!);) spread ctolloidal magnetic iron on p o l i d i d .-\hiic.o 5’ alloy and investigstetl the rcsulting liarticulate patterns during tlemagnetizntion of the solid Mnico magnet. Still another shape of particle for metallographic research i? the “uvhisker.” Scott and Coleman (60) studied the deformation and corrosion of iron whiskers, and Laukonis and Coleman (10;j studied t h r morphological changes on specific crystallographic faces of a-iron whiskers duririg oxidation. Takagi (172) and Hashimoto, Tannka, Yoda, and . h k i (8:) studied the oxidation of tungsten filaments. The bulk of electron nietallographir* work, hovc-evrr. is still accomplished by replicating the prrparcd surface of hand specimens. Thc -1STJI S u b committee XI of E-4 on Elwtron IIicrostructure of lletnls continues to lead in technique, information. and commimication. It has recently reported ( 1 1 ) 100 pages of exccllrnt work. with over 90 beautiful reproductions of esemplnry micrographs of typical appearances as a result of thoroughly tested procdurrs. Thr trchniqups cniployed are suniniarized clearly. Electron microscop>- appears t o be advanta98 R
ANALYTICAL CHEMISTRY
geous in depicting the roughening of retained p-tit’ariium during subcritical aging but prior to formation of resolvable alpha or omega particles and for other phase relationships and testures. Young and Schwartz (60) extend such studies by straining Folishcd and etched samples of titanium alloys and replicating thcni a t various stages of deformntion. The symposium on advances in c,lectron metallography, sponsored by -%ST11 Subcommittee XI of E-1 a t Boston, June 1958. has been published ( 9 ) together n.ith an additional paper, on selected etchants for electron niic~oscopical studies of niagnesiuni alloys by Noe. --\mong the papers on technique Long and Gar>-report on vibratory polishing of specimens, and l3ridgi.s and Long discws the e:is\. rcniov:il of replicas. JIucii work is hcing done on Iinnd specinicns of -ingle (ai.yit:ilswith rp3pei.t t o thiir ywcifii. f : i i w . Soggle :tnd Ytiegkr (138) :tiitlicd i~hcniirnllyetched f:i(.r? of gc>rniariiuni Iic~Fort~arid :iftvr irradiation with ncwtron. ,\ l i i k Grwnd v e r and ficld (.TO) thcrnis!l>. t~t‘~iii~i1 found that iiidcx f:ii>i+ (11 1 .ind !IO) wcre prcferc.n t in 11y i l < ~ \ - c ~1.l o ~ ) ~ ~ ( E:vcri t u:il ly (~saiiiina t ion iiiuat carrim1 i,tit on ni:isR-!)roilucc.il coninicrcia1 nititvriak, t o coiitrol I)ro(iuc.tion or to rleti~rminc f:iilun%. Srhn.:irtz :inti Young ( 1 6 1 ) iic~vchqicii tt>i,tinicqws for !ionclr,itriic’ti.,.c ic;,lii.:ition of mcmtal surfacc~s niodific3cl I)?. h i r i n g . i\.c?ir, rorroiion, : i i i ( i fatigiip olitsiilt, tlw iahorator!.. Thew maatcr rt>plir:is C ~ I I he furtlicr rt~pri)duud so :!]at !)(it!: elertrc,n n n d light nlic.rci~r,i) carritd out on the s2m> :II’ a n d Grutw i.50) riv.c,ntl\. rclporteii on tht.ir careful stud>- of carhurizd or carbonitridid steel.. with rr.l:ition to f:itiguix and rwiriuai strr.sscbs. Of utmost niet:illurgic~:tl inii)ortance is the furthc’r understanding of hurtlcning by precipitation i n nl1o.r-s. I’itsi.11 ant1 .\iig(~li(~:i(.;I) :ind C‘ai;i,ncis :ind Hnurer (:ill li:i~.c rewntl>. 5tuditd the niorphological and structural i,liaiigc* at critical tcmper:iturc~iin :ilioycd stt’C1‘. For tho past 30 years aluminum has been added to tleosidizc stwl. but tllca fate and bi,havior of tlit, c’xiws aluiiiiniim have not been iintlcrstood. ( w e p t that it formed tht. nitride if possihlc. Hsiao ((5’8) has bwn able ti) iilciitify (by clcctron tiiffr:rction i anti thryn r c i q nizc ( b y clcctron niic~roscopy)the, till). pnrticlw i100 to YO0 A , ) of aluminum (111) riitritlc distributed in situ. The ositiation of preparcd spccimena may be experiinent:iIl>. useful for a number of piirp(iw. K r i ~ l i i ~ n h t ~and rg Tenzcr (101i simply allon.c,c! wide films to form naturally on elcctropolished .%lnico V alloy and examined the rciultant films as replicas. On thr, other hand, Hsino ( S i ) foinir! that tht, oside film on hot-dipped tin p h t e w:is mior-
phous, so he bombarded the film with the electron beam of the microscope. -1 particulate morphology and character.istic diffraction pattern were then obtained. Miller and Lawless (121) cheniically osidized specific faces of a large single crystal of pure copper. Replicas of all of the osidized faces depicted crystals of copper(1) oxide! which onl\on their 111 faces showed a spiral growth, evidently controlled by dislocations. -\llison and Samelson (.5) used oxide-film formations to study the diffusion rates of aluminum. magnesium, silicon. or zirconium added ningly to nickel. Generally the corrosion resistance of stainless ateel was attributed to the effective esistence of substantial oxide film on the exposed surface. Rowe. JIt!w. Sicolayien, and Rigga ( 7 ) , elwtron diffraction. did identify crystalline c~hromiuni(I1I)oside-nickel (11i olide iCrr03 SiO)on thr surfaw of ty!iic:il st:iinles steels. .\ftcr r w w n on, however. the!- coulti finti no rvi’. ?eemirigl!- c omcs froni rv)nihining terms like , ,aniorphow i.r?stal>“ 2nd ccinfiising definite ((10)ternis “ ~ ~ r ~ , ~ ~ t a l ln.ith i n i t y‘acr~,~stal!iz:i” ,fnrni.” “face.” “habit.” and ‘,plin+” n-ith one another. Yo di4ncation is mitie lwt\vwn spoiitancoris crJ.5:a1 Krc;n-th scsif-containid iorws :intl in!an-nia(i? ani-otrop!. 5uc.h a ? h!. strc,tchiiiq ior roiling. Itoreover. c>lec.tron anti x - r n ~ .iliffrartion y t t c m ; arc :~.>iinwiI to be tiir mlt, ;.riterioii !‘or c.r!-.t:iliinit!.. .with :ii-rr.gsrti for more \vitlistsntiing (.riteria su(~1ia ? goniometrj,. optic:ii propcrtics. light aiid cleci r o n intc.ricrt~nc~e p:ittiJrns, a n d . most of 311. t)t’!ia\.i(Jr during gro\vtli. i ii,lorniation. :nc,ltiiig. : i n i i dissolution. Keith (I&?), P:iildt,n i ! {i‘i. m i l .\Iorc1ic’ad (1 4.7) anti their colleagut~; ha\^ 1)wn1t~:itIc’rc.in ;,onihinirig cla&:il light microscop!. ivith electron niicrowopy if: cscrlleiit studies a n d scholarly prwrritations of the growth, structure, !labit, and drform:ition of cr~.stalline polJ-eth! ltlnc nnd polypropylene. Keith, Puilden. V7altrr. a n d W!.ckofl’ (94) explaincd some micromorpholggicul differences in terms of s-ray diffraction :11id concluded that besides the nionol isotactic pol>.prop! icne clinic, p h : ~ ~ c of ( t ,?I)there is a !iex;igonal phase.
Geil, Symons, and Scott (63) have also performed difficult work using complementary methods. They grew single crystals of polyoxyniethylene : six-sided platelets with screw dislocations displaying moire lines by dark-field electron microscopy. Electron diffraction indicated that the chain molecules are normal t o the plane of the platelets and are folded. Geil (145) also concurs in chain folding and lamellae of single crystals for several polyamides. Eppe, Fischer, and Stuart (52) obtained single crystals of low pressure polyethylene from solution and according t o electron diffraction the molecular chains are normal t o the lamellar plane or fibrillar direction. By both electron microscopy and low-angle x-ray scattering they showed that polyurethane could be obtained either crystallized or amorphous by slowly cooling or quenching, respectively, from the glassy state. This is a departure from the imaginative picturization which formerly prevailed. Keller (55-97) has reviewed the electron microscopy of crystalline high polymers. He interprets a deterioration of the electron diffraction pattern of nylons during the electron bombardment as a destruction of crystallinity (57). Little (57) observed the same phenomenon, but Keller acknowledged the communication as a claim that such irradiated specimens could still be classified as crystalline on the basis of other properties. Kargin and Koretskaya (93) lost electron diffraction patterns by irradiating polyamides or polyethylene in the electron microscope. Agar, Frank, and Keller (1) have succeeded in obtaining electron micrographs of polyethylene without deteriorating the electron diffraction pattern. Siegisch (51) does not mention a deteriorating effect in obtaining his electron diffraction patterns and niicrographs of polyethylene. I n contradiction with Kargin and Koretskaya (M), Siegisch (5f) illustrates the close relation between simple crystals and spherulites. Holland (145) prepared classical spherulites of polyacrylonitrile from solution in dimethylformamide, measured some light optical properties, and depicted light and electron microscopic morphology. Miller and Rauhut (122) prepared poly(tert-butyl acrylate) , part of which manifested light microscopic spherulites and part displayed a n electron microscopic skeletal habit. Uurge (34)slowly evaporated aqueous solutions of poly->proline 11 (average degree bf polymerization 20, average chain length 60 4.) and obtained wedgeshaped single crystals. These displayed interference fringes which he interpreted t o be related t o the double refraction within the crystals. Amorphous Polymers. Like crystalline polymers, amorphous polymers
are constructed and behave as though they a r e composed of several classes of microscopic units (96). But the macromolecule, itself, appears t o be the most interesting and definitely is the most difficult t o depict. Nevertheless, in microbiochemistry a t least, there has been general acceptance that the giant macromolecules of certain proteins may be isolated and depicted with fidelity. If there remained any doubt, Hall and Doty (71) have removed it by their improved technique, careful choice of substances, and plenty of independent data. These experts confirmed the long shapes and expected average diameters, size distribution, and macromolecular weights for their samples: ichthyocol collagen, deoxyribose nucleic acid, poly-bglutonic acid. Their results were in very good agreement with the accepted indirect values obtained by flow birefringence, viscosity, lightscattering, and sedimentation rate. The direct observation helped explain what variations there were in the indirectly obtained data. Moreover the beaded shape and low degrees of winding and intertwining were observed. Hodge and Schmitt (84)examined the highly ordered aggregates regenerated from collagen solution by sonic vibrations. Their electron micrographs of very high resolution and contrast show a fascinating degree of order, especially when compared with the excellent work of Kuhn, Grassmann, and Hofmann (102) on stained natural collagen. Semetschek (134) has continued to study the morphology of natural collagen, the cross striations, elementary fibrils, and order within the cells. Referring t o the method of Backus and Williams ( I 7 ) , Koretskaya and Karpov (95) tried t o reduce the chance for aggregation of macromolecules, which may occur during evaporation of the solvent. They pipet the solvent away, after some macromolecules have been absorbed on the nitrocellulose substrate. Their electron micrographs of thallium polyacrylate and of polymethacrylate, however, show a much higher concentration of macromolecules than was in their dilute solution; therefore the question df aggregation could remain. Frajola and Greider (50) formed a monolayer of deoxyribonucleic acid (DXA) in a Langmuir trough equipped with a device for inserting and removing a piece of freshly cleaned mica t o which DSA adhered. They qualitatively compared their technique with t h a t of Williams and Backus (17). Whether the former includes quantitative aspects is not known from only the published abstract. There is also the intriguing abstract by Heyn (145) who studied acrylonitrile macromolecules, microgel, and larger particles of irregular shape. Thomas, Thomas, and Deichert ( I 76) discussed a microscopical study of
vinyl polymers and further established Botty’s classification of particles (I, macromolecules; 11, colloidal particles; 111, their cluster; IV, discrete aggregates of I1 or 111). They studied the steps in heterogeneous polymerization of acrylamide, acrylonitrile, vinyl chloride, vinylidene chloride, and chlorotrifluoroethylene. The growth steps in each polymer were examined; the results provided a missing part of previous studies of polymerization mechanism. Sagao and Uchida (130)studied steps in the aqueous polymerization of acrylonitrile and found that salt addition and p H of the medium greatly affect the appearance and formulation of particles as well as the rate and degree of ,polymerization. .After a slow start a decade ago (155) the examination of the fundamental units in situ is gaining more recognition. Moore and Peck (124) investigated highly branched polyethylene in 1%hich light-scattering indicated the presence of very large particles. They show evidence that the particles are not foreign material, microgel, or aggregates of molecules. Electron microscopy of toluene-etched cast films depicted round particles, as expected, of highly branched macromolecules. This morphology, together \+ith solubility and behavior on pyrolysis, strongly indicates that the particles are single, very large macromolecules (500 A. in diameter; 40,000,000 molecular weight). If there are macromolecular boundaries, then by classical definition, the smaller depicted units must also be macromolecular, but ?tloore and Peck (f24) do not declare this. They do emphasize the advantages of examining macromolecules in situ by remarking that their electron microscopical measurements of diameter are smaller than those indicated by light scattering. They explain the disagreement on the basis of expected swelling by any solvents. I n support of this explanation is the work of Burnett, Lehrle, Ovenall, and Peaker with polystyrene latices (35). They found good agreement between number of particles based on light scattering, electron microscopical count, and measurement of diameters. Erath and Spurr (63) compared fractography (155, 157) with other preparatory techniques in their study of phenolic, diallyl phthalate, epoxy, and silicone resins in order to understand why phenolic resins, for example, had a measured tensile strength which is only one fifth of t h a t calculated on a theoretical basis of van der Waals forces. They observed that fracture occurs between the globular formations (155) of colloidal sizes, which they call micelles. They compare the linear tendency toward arrangement of the micelles with filaments observed in the phenolic resin in an early stage of curing. They do not VOL. 32, NO. 5, APRIL 1960
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name or otherwise classify the abundant units which are smaller than micelles, especially in silicone resins (154). Andrews and Walsh (15) extend the application of the earlier methods (154) t o prepare surfaces of brittle fracture and slow tear in various samples of rubbers and carbon black t o explain the functions and behavior of reinforcing agents. Their confidence in the structurelessness of evaporated carbon used to replicate the location of carbon black particles is well founded. Fischer (58) studied very extensively the stability of pigments suspended in solutions of high polymers uhich are used in surface coatings. He included the effect of additives such as polyelectrolytes on latex particles of polystyrene. The electron microscopy and micrography are excellent. Weissbein and Coven (188) studied the electron microscopic distribution of direct dyes and its infIuence on the light fastness of colored films and fibers of regenerated cellulose. They found that certain direct dyes exist a t least partly in a n aggregated state which is important to light fastness of the colored material. Fibers. Whereas wool (61) is t h e chief natural fiber of intefest t o t h e British, natural, modified, and regenerated cellulose is of chief concern to the United States. Tripp, Moore, deGruy, and Rollins ( 1 4 , 156), of the Southern Regional Research Laboratory, continue to lead in the electron microscopy of cotton. They use replicas, ultrathin sections, fragments, and on problems oi other techniques to ~ o r k dyeing, mercerization, hydrolysis, resin impregnation, and derivatives of cellulose. Cot6 and Day (50) are studying wool and prefer using replicas or pseudo replicas rather than ultrathin sections. Honjo and Katanabe (86), using a cold , studying the morphology stage ( E ) are and structure of cellulose employing valonia (153) fibrils. They show by electron micrographs and diffraction patterns that morphology and structure may be preserved during the electron bombardment by cooling to -40“ C. or below. The arrangement of cellobiose chains is different from that proposed for cellulose-I by Jfeyer and Misch in 1937; Honjo and Watanabe (86) are not yet certain whether they have shown a new structure or the correct one for cellulose-I. Besides viscose, Scott ( 1 4 )is studying several other kinds of synthetic fibers. The fiber is cut part way and stripped off the longitudinal section of the skin or core. A thick section is etched, for example, with n-propylaniine for Dacron or other polyesters. Anderson and Holland (51) are studying nylon fibers by embrittling and fracturing them. Botty, Anderson, and Felton (145) are studying acrylic fibers by light and electron microscopy and microradiogra100 R
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ANALYnCAL CHEMISTRY
phy. Differences in structure were demonstrated in cross section and by replication techniques as well as by examination of the bulk fiber. I n another area of protein chemistry, Fraser, Macrae, and Rogers (61) are studying the structure of a-keratin in developing hairs and degraded wool. They combined high resolution of electron microscopy with x-ray diffraction and infrared dichroism. They and Simpson and Sikorski (50) are concerned with specific staining of the sulfur of keratin in hairs. BIOLOGY
Applications t o biological problems continue to consume over half the publications and presentations on electron microscopy. The many long years of perfecting techniques and interpretations are now showing greater rewards. The first of these was pure description of static specimens which has not only a n intrinsic value but, as Dalton said (51), it is the starting point of experimental analyses which Denues, hlothram, and Biesele (50) termed “functional studies of dynamic processes.” They used the punched cards of the XYSEM Bibliography (153) and found that the ratio of dynamic studies to static studies increased from 1 to 4 in 1950 to 1 t o 2 in 1956. From these data and other library research, they see the timeliness in current research and further need in program planning for increased emphasis on functional studies. This appears t o be the urgent need if we are to gain the full fruit of recent technical developments for advances, including those against cancer. The extensive symposia on cancer and viruses ( 5 1 ) , jointly supported by the American Cancer Society, Inc., and EMSX a t Columbus, Ohio, 1959, were well received. Dalton, Friend, and Mottram (51) xere able to identify the true tumor cell capable of inducing leukemia in mice. Previously deHarven and Friend (47) had observed virus particles in leukemic mice which were not observed in control animals. Dmochowski (51) iliustrated the relationship of viruses to spontaneous and- x-ray induced leukemia. Beard (51) summarized the role of electron microscopy ’ in characterizing canecr causing \’iruses. Swift and Rasch (171) and Morgan, Rose, and Moore (125) have reviexed electron microscopical techniques, descriptions, and functional studies in cytochemistry and studies of intracellular virus> respectively. Smith end Killisms (165) used a clever shadoxcasting technique to show the exact geometric form of Tipula irridescent virus particles. Lauffer. Ansevin, Cartwright, and Brinton (103) reported the reversibility of the polymerization process of tobacco mosaic virus protein, while Fernhdez-MorAn and Schramm (57) showed its fine structure by ob-
taining high resolution micrographs of stained ultrathin sections. Dmochowski, Grey, and Burmester (50) also used ultrathin sections to observe the possible life cycle of certain viruses. Nylen and Scott (139) have composed a’ monograph on dentinogenesis. FernAndez-MorAn (55)extensively reviewed the nervous system; Yssuzumi and Deguchi (196), Rolken ( 1 9 3 , Kissen and Craig (51), and Tousimis and Fine (51) have investigated the structure of the compound eye. Significant progress by Huxley interpreting the action of muscles was made by investigating the interrelating structural units (90). Further studies have been reported by Bessis and Breton-Gorius (25), Richter (50), and Farquhar and Palade (51) on the functions of ferritin in various systems. Preston’s review (148) is a n excellent summary of the part played by electron microscopists in the recent developments of the biological sciences. During the AAAS meeting in Chicago, December 1959, there was an all-day symposium on “The Impact of Electron hlicroscopy on Biology.” X-RAY MICROANALYSIS AND IMAGERY
One of the most challenging and potentially rewarding uses of a n electron microbeam is t o analyze qualitatively and quantitatively for the chemical elements in situ in microsamples. The microbeam is obtained by a condenser such as is used in the electron-lens microscope; a n image of the source of electrons is demagnified to a diameter varying among instruments from 100 to only several angstrom units. X-rays are generated by the specimen in the emission method and a t a selected metal target in the absorption method so that by characteristic fluorescence or by preferential absorption a specimen can be analyzed. I n any case, the apparatus is still elaborate and expensive. The emission technique (electron probe), originated by Castaing (153), is the chief one and some 10 such instruments. with either fixed or scanning probes, have been hand-built here and abroad. Fisher (51) has discussed the problenis involved. Commercial availability has begun (153). Canalco considers all techniques to be desirable and is proriding such versatile apparatus. Typical applications n-ere discussed a t the Second International Symposium on X-Ray llicroscopy and X-Ray XIicroanalysis a t Stockholm, June 1959 (1%). -4typical study is the diffusion of alloying elements n-ithin grains and along grain boundaries. Some purposes are to study mutual diffusion during casting and annealing processes, segregation (9), and inclusion, especially with respect to fracture and corrosion. I n addition (51) there have been applications in the study of minerals, bones, paper, and storing information in microspace (50).
A short comprehensive reviGw of microscopy with x-rays by Cosslett (44) offers a useful summary of the several x-ray methods that can be applied to the study of materials for their subsurface textures as well as for the microanalysis of many of their atomic constituents. For more detail the reader is referred to the published proceedings of the 1956 symposium a t Cambridge (194), and the forthcoming proceedings of the 1959 symposium a t Stockholm (195) on x-ray microscopy and allied subjects, which in addition to reviewing the prevailing state of the instrumental technology, also includes numerous examples of applications of this somewhat neglected method of instrumentation. In x-ray imagery Foley and Kewt m r y ( % ) , using a General Electric Type A5001 shadow x-ray microscope, studied stress corrosion cracking of a magnesium-aluminum alloy and pitting corrosion of three different aluminum alloys. Their significant contribution indicates how x-ray microscopy permits the investigation of composition and structure of the surface as well as reactions within the alloy. X-ray micrographs show that the development of etch pits and the propagation of cracks due to stress corrosion varied in differently treated magnesium-aluminum alloys. The apparent utility of these methods should suggest numerous critical experiments to those who study inorganic, organic, and biologica! systems. With the proper selection of reagents, one should be able to detect diffrrent phases and centers of reactivity within materials t h a t are normally opaque to light and electrons. Bessen (50) outlined three analytical methods applicable with the K'orelco x-ray microscope and emphasized that the use of each method requires the proper selection and adjustment of the x-ray spectra. Of the three, absorption spectrochemical analysis should be of most interest to the analytical chemist. Uy taking advantage of the great depths of field of the shadow projection x-ray microscope to obtain stereoscopic vieivs, Saunders and Frj-e (50) have investigated the vascular patterns associdted with certain parts of the brain and spinal cord. They appear to place considerable emphasis on the potentialities of shadow projection of x-rays. Prrliminary test films of stereo x-ray movics were made by Xorton and ?;en-berry (50). By shifting the electron spot between exposures and keeping the sptbcinien stationary they recorded left and right images on alternate frames. Although they claim that no simple method for modifying standard units of spot deflection exists, and that stereo projection necessitates highly specialized equipment, the fact that a start has been made is commendable.
hIany of the principles which apply t o x-ray microscopy also apply to microradiography. Botty and Thomas (61) modified a conventional electron microscope for use as a microradiographic instrument and showed the advantages and limitations of their method when applied t o various polyphase materials. Niskanen (136) used a Norelco x-ray spectrograph for selecting the desired monochromatic beam of x-rays to enhance contrast and depict specific metal-rich constituents in a variety of specimens. H e made full use of the critical absorption of the elements present to obtain very satisfactory results. Although his method sometimes requires long exposures because of the relatively low intensity of the monochromatic x-rays, this method appears to be justified where distribution studies are to be made on complex materials. TWORCA Stereo-Microradiography cameras were designed as attachments to the RCA x-ray diffraction instrument. These apparently versatile cameras permit multiple microradiography of small objects and thin sections for internal structure, micro defects (I3,!i?), and thickness measurments. Their Splettstosser type camera utilizes the direct beam from the x-ray diffraction tube to excite a secondary target of a selected metal, thereby forming a characteristic fluorescence emission which produces the microradiograph. Thus by proper selection of monochromatic radiation a specific element can be made to absorb the radiation selectively and be depicted in high contrast and detail. I n some instances the somewhat higher s-ray intensities frequently ob tainable in certain microradiographic devices is a distinct advantage. Pattee (144) constructed a microfluoroscope for the direct viewing of materials 1 to 10 microns thick. This clever instrument which resembles a medical x-ray fluoroscope in many aspects, captures the highly intense image of the specimen on a fluorescent screen, 0.5 micron thick. The fluorescent image can be further studied with an auxiliary light microscope and, if desired, with photometric s-ray absorption equipment. The optical principles, the method of construction, and the comparative photographs of light and x-ray images of test specimens were clearly presented. FIELD
EMISSION MICROSCOPY
During the session on microscopical specializations of the -4CS sjmposiuni in Boston ( 7 ) , April 1959. LIuller, originator of field emission microscopy, said that the field emission microscope is a very specialized instrument with many limitations. However, in the form of the field electron microscope, it has shown its usefulness for studling adsorption layers on metal crystals in a
temperature range from almost 0' to 2000" K., and has contributed to the concept of clean surfaces. I n 'the form of the low temperature field ion microscope i t is the most powerful of all microscopes, with a resolution approaching 2.3 A. and the unique capability of showing the individual atoms that constitute the lattice in the surface of a crystal. The arrangement of atoms in imperfect lattices such as dislocations or stacking faults as well as disorders of originally perfect structures due to chemical corrosion, fatigue, or radiation damage can be seen directly. Up to this biennium, field emission microscopy was limited to the study of platinum, tungsten, their close relatives, and their compounds (153), but recently Gomer (66, 67) grew whiskers of mercury from its vapor in the field emission microscope, The whiskers were so satisfactory as cold points of field-electron or ion emission that hIelmed and Gomer (117) tried to make useful points of other metals in the same way. I n the field emission tube a heatable wire (substrate) of tungsten is looped with a separate tungsten wire which is wrapped with the sample to be evaporated. The loop and sample are made hotter than the substrate to give about mm. vapor pressure of sample and thereby grow whiskers in situ in 5 to 10 minutes. I n this way whiskers were grown of aluminum, copper, iron, nickel, silicon, molybdenum, and carbon, besides mercury already mentioned. The resultant points were useful in field electronemission and also gave promise of being useful in ion-emission microscopy. I n the meantime, Cooper and 3Iuller (42) improved on the original 3Iiiler technique for the study of adsorption and other phenomena on the surface of metals besides the platinum and tungsten families. They obtainrd clean surfaces of conventional tips from wires of softer, more reactive metals such as nickel, iron, and copper, without heating. They devised an alternating field technique with a de-biased alternating current power supply for providing a desorption field during the positive portion of the cycle and allowing the observation of a field electron image during the negative portion of the cycle. Desorption data with respect to orystallographic orientation on cold-worked, unannealed metals indicate that more information will also be obtained on t h e platinum and tungsten types of pure metals. So far, Cooper and lluller have n.orked on only a few systems: the old one of barium on tungsten. also tellurium on tungsten, and nitrogen on iridium, among the number of possibilities opened up by the nek technique, By the older conventional procedures alone, field emission microscop)- has grown fast in its applications (4, 30, 46, 118). However, the whisker technique VOL. 32, NO. 5, APRIL 1960
101 R
of Gomer and Melmed and the alter nating field technique of Muller and Cooper represent major means for breaking through to the study of specific atoms in most metals, metalloids, and their systems, especially in alloying, distortion, annealing, corrosion, and catalysis. SUMMARY AND CONCLUSIONS
The intention of these reviewers is to select critically some recent electron microscopical advances and to criticize constructively. We are b u t one small team with only a limited time. Inadvertently or through necessity many interesting and useful contributions were omitted from the text and bibliography. In the past biennium electron microscopy has been integrated with, rather than differentiated from, light microscopy. While adding to the knowledge of micromorphology, electron microscopy is also contributing to the understanding of physical-chemical analyses, syntheses, and behavior. Some of the methods are of classical chemistry 01‘ physics and are inexpensive. Others require elaborate apparatus, such as the electron probe, and are indicated for highly repetitive analyses such as are required in metallurgy. Other methods, such as Miller’s field microscopy, are still being developed for a promising future. I n all of this, electron microscopy has advanced from proving that a biological, chemical, or physical unit exists to studying and controlling the behavior of the whole material in terms of its units. l l o k t optimistic of all is the trend toward demonstrating action and reaction inside the electron microscope. ACKNOWLEDGMENT
Again we acknodedge the aid from the staff of the Research Laboratories of American Cyanamid Co., the Technical Information Section, and oGr secretary, R. J. Verrilli. This biennium we have had the privilege of receiving constructive criticism of our first review, a n d we have tried to use the advice. We heartily invite comments from our current readers. LITERATURE CITED
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