Electron Microscopy M . C. Botty, American Cyanamid Co., Stamford, Conn., M . C. Davies, lederle laboratories Division, American Cyanamid Co., Pearl River, N. Y., and C. D. Felton, FMC Corporation, American Viscose Division, Marcus Hook, Pa.
T
covei-s the period from January 1962 to January 1964, a two-year interval that has included remarkable progress iri electron microscopy as well as in otk er scientific areas. The concurrent growth of the many aspects of electron microscopy, which is reflected by improvements in instrumentation, development of new methods of specimen preparation and a n increase in the number of microscopes and microscopists, has resulted in extensive investipation of a wide variety of subjects. The electron microscope is a u g m n t i n g the more usual methods of diagnostic surgical pathology, sometimes on a routine basis (242); a t the other extreme, the morphology and composition of meteoric dust has been studied (190, 424). Although the electron microscope is sometimes alluded :o as just another tool, the abundance of literature describing fruitful applications of this instrument emphasizes its indispensable role in providing for a better understanding of the organization of matter. This in turn has contributed to the solution of many industrial and biological problems which otherwise would have required indirect and timeconsuming methods of investigation to achieve comparable rcsults. I n a critical and contemplative evaluation of the -Jresent state of scientific activity, Hubbert (215) calls attention to the fact t>hatthe number of scientific papers has just about doubled in the past 15 years. The rate at which scientific papers are being published is accelerating. Chemical Abstracts has published ’ t s three millionth abstract: the first abstract was published in 1907, the first million 32 years later, the second million 17 years later, and the third million after only 8 more years (81). It appear,j that part of this voluminous expansion of the literature may be due in part t o sutomation in the laboratory. The vwious types of laboratory procedure:, that are amenable to automation, thereby augmenting scientific activity by making more time available for creative kvork, is discussed by Picard (360). He considers microscopy, which is an a r t as well as a n interpretive discipline, to be primarily outside the realm of automation. Kevertheless, microscopy continues to grow and to permeate the diverse scientific literature. :In the absence of HIS REVIEW
a journal of electron microscopy in the English language, the prime purpose of this review is t o critically consider the significant contributions and trends in microscopy as they are associated with general and specialized areas of scientific activity. I n some instances our endeavors are eased by the inclusion of well-referenced papers which in themselves constitute reviews on specific subjects. I n other cases, extended coverage is necessary because the markedly increased activity in particular scientific fields gives rise t o a considerable amount of new and important information. This review is primarily confined to papers that have been published in the English language, although it is clearly evident from the two-volume, preprinted proceedings of the Fifth International Congress for Electron Microscopy (ZdO), containing over 600 papers, that electron microscopists are active on a world-wide scale. INSTRUMENTS
The electron microscopes of today had their origin in the discovery of the cathode ray tube and in its subsequent modification and adaptation by electrical engineers for making more accurate measurements of high frequency oscillations. The somewhat erratic course of progress leading to the first production model in Europe, the Siemens’ electron microscope of 1939, is described by Mulvey (319). This informative paper contains a bibliography of many who participated in terms of practical thought and deed toward the realization of a functional instrument. I n a related paper, Freundlich (150) accents the important discoveries by scientists in the decade after 1920 which accelerated development. He calls attention to the significant modification of the magnetic lens by Knoll and Ruska which gave rise t o a two-lens experimental prototype electron microscope in 1931 and to a three-lens model in 1933 which had a resolving power better than existing light microscopes. Recognizing Mulvey’s excellent historical treatment of the origin and development of the electron microscope (319), Freundlich emphasizes that although Knoll and Ruska were the first to advantageously reduce to practice their concepts of electron optics, thus producing a working electron microscope,
they were not, the first to apply for covering patents. Porter (369), in published preliminary greetings to the delegates of the Fifth International Congress for Electron hIicroxopy, briefly summarizes circumstances which favored the development and practice of electron microscopy on this continent in the years following 1940. Cocks, hlelton and Schwartz (BO) review the different kinds of instruments that qualify as electron microscopes and t,he various techniques and conditions that produce useful microscopical images. The versatility of the instrument, its shortcomings and potential, ab well as the need for experienced personnel, are thoughtfully presented. In another paper, which includes schematic drawings, Cocks (89) discusses the functioning of the electron microscope and the attempts that are being made to improve performance. Gilmore (158) describes transmission and ion emission microscopes in considerable detail and also points t o the advances in electron microscopy that promise to extend the resolving power of the instrument. Regarding low voltage electron microscopy, he mentions Wilska’s new lens design which provides for a narrow annular slit situated within the lens. Only electrons in the peripheral part of the beam can pass through the lens. This reduces spherical aberrations while still maintaining the beneficial, high image-contrast which is produced when thin specimens are examined with relatively slow-moving electrons. Interest in one of Kilska’s microscopes has made news (350) in that a resolving power of 2 A. was forecast. I t is anticipated that further $ e v e l o p e n t and applications of low voltage instruments will make n-ell-remembered contributions to the history of electron microscopy. Wilaka has reported (220) on the design and construction of one of his electron microscopes which has a working range of 1 to 50 kv. A h m i g other features, this instrument is equipped with a device for heating the objective aperture by nieans ( J f a direct current in order to keep the a p c ~ t u r e clean while the instrument is in u i e . h cleaning device for removing sipertiircb contamination has been built into a ( Y I I I I mercial electron microscope. the A k a ~ t i i Tronscope 80. I n most microscopes the rate of contamination can he quite ra1)i(l. VOL. 3 6 , NO. 5 , APRIL 1964
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Under controlled conditions, Patterson and \Vayniun (351) measured the rates of contamination visible a t the edge of niol!~btlrnum oxide c r p t a l s in an elevtron niic*ro;.cope operated a t 100 kv. Orie of these conditions involved the UT of st c-lccontaniination apparatus of their o \ v n tie-ign which condensed nndehirable hydrocwbon vapors on a metal surfare that was cooled with liquid nitrogiln in rlie vicinity of t,he specimen. \Yith the caooling device. the contaminat,ion rate Ira. found to be 0.48 AJsec.; without it, the rate \vas 1.11 -1 a further advantage this dev used in ronjunction with accessories such as the multiaxi.; tilting stage and tensile straining devices. .1n apparatus having a similar function is now supplied with the most recent HV-11.1, high resolution electron microscope, manufactured b y Hitachi Ltd. Heide (220) has also experimented with a very efficient cooling device. Dupou>-. Perrier and I h r r i e u (133)report on the rate of contamination of LIgO crystals in air arid in vacuuni a t voltages between ld-85 liv. Specimen chambers for esarnination of the specimen in a gasecius atmosphere are described. R’hen the specimen was surrounded by air. the rate of contamination was markedly reduced. Any instrunientation that alleviates the serious problem of contamination is of value to the micros:c’ol)ist, The specimen holder may bevome contaminated by handling it with the fingers and subsequent contamination of the specimen may occur, To avoid this. Pangborn (546) has devizcd a c2aiq)ing device for the standard RC.1 s p e h i e n holder. Specimen contamination of a more serious nature may result from condensation of diffusion or fore pump oil. Ion pumps such as are used in some vacuum evallorators, offer freedom from contamination of this kind. The principles of oileration and the advantages associated with ion pumps have been discussed ( 1 7 2 , 398). Perhaps manufacturers of electron microscopes will
--
u t ilizcd. I~:lertron micLroscopes now cover the \\.orking rangi’ from 1 t o 1000 k v . l,;:irl>, invwtig:ators were n-ell aware of thc, infliic>nc-cof lo^ beam ilotential on rnlianc,ing image (‘ontra*t. Van Dorhten atid I’rcnwla ($4) rcxviewed this subjert ant1 i)rovidcd gral)hic evidence of t h c vnlucs of lo\\. voltage microscopy from tlit,ir o\vn eslmimentation as aliljlitd t o ultra-thin sections of I,ioloeic>:ilti-zuc l r w than 100 -1.thick. (-5ing a :13-niirron objective aljerture. t h v i r iiiiI)rcshiv. iiiwriing a -mall, tubular c ~ ) i i i 1 i o n ( ~ 1 1in1 1 o t l i e anode aiierture of tlir l ’ l i i l i l ~ ~I,:lI-i513, I3y decreasing 174 R
ANALYTICAL CHEMISTRY
the distance from the end of the tube to the tip of the filament, good gun efficiency was maintained a t 13 kv. and well resolved, high contrast micrographs ivere obtained. Considerable care was taken to remove contamination from the inside of the column and to select photographic material which would have suitable gamma at lon. voltxges. The construction and performance of the 300 kv. electron microscope a t Kyoto Ilniversity, Japan, is discussed by Kobayashi et al. (243). The resolving power of this instrument was demonstrated to be 20 A. a t 300 kv., using the granular detail visible in a Pt-Pd vacuum-deposited film as a test sample. The transmishive power of the electron beam varies with the test specimen and the voltage. The spacing of thickness fringes in a wedge-shaped LIgO platelet ihowed that the beam penetrated through a crystal that \vas at least 1.5 microns thick. In an electroliolihhed stainless steel film, fringes due to stacking faults indicated a thickness of about 8400 -1. The degrading effect of electron irradiation on the >tructure of a polyethylene film was demonstrated by changes visible in a sequence of electron diffraction patterns. Dupouy and Perrier (152, 220) provide a detailed description of the 1000 kv. electron microscope which is functioning daily at a laboratory in Toulouse, France. This instrument has been applied to the ytudy of metals 2 t o 3 microns thick. ’They a h o reported that living bacteria. which- were contained in a special, air-filled specimen chamber, survived this high-energy exposure. In their laboratory, materials have been examined in brightfield and darkfield, and well-resolved moiri. and electron diffraction patterns have been obtained and evaluated. Most commercial electron microscopes in current, upe operate between 50 and 100 kv. and are sold a t a price which increases with improved resolving power and versatility. The qJ13-30 ( I @ ) , manufactured by the Japan Electron Optics Laboratory Co., Ltd., and the ELI-20 (5S), available from Mikros, Inc.>are new commercial instruments that operate below 50 kv. ‘The cost of these moderately-lriced instruments also varies with their respective resolving 1)ower. I?niver$ities and control laboratories will find these instruments attractive for many routine electron microscopical examination?. especially when the ultimate in resolving power associated with the more poiverful, but more csliensive newer instruments, is not required or is not economically feasible. Microscopists who attended the Fifth International Congress for Electron hlicmscol)y in 1962 had an opportunity to operate or witness demonstrations of
electron microscopes and accessories made available by approximately twenty-nine commercial exhibitors. In recognition of their significant contributions to the growth of electron microscopy, through the quality, versatility and continuing development of their products, there is a desire to give each product descriptive coverage. This, of course, is not within the scope of this review. Fortunately, some of the equipment has been described elsewhere (8, 38, 378). In addition, excellent descriptive literature, which frequently includes schematic diagrams, micrographs, sectioning techniques and other useful information, is available on request from the manufacturers or distributors who advertise in many of the journals contained in the bibliography of this review-. I5gelow (58) reviews the developments of transmission elect,ron microscopes for use in metallurgical work, and then describes the development of reflection, scanning, mirror, emission and point projection microscopes which are less frequently used but which permit effective, direct examination of the surface of metallurgical specimens. He cites numerous examples of application and includes a chart which lists in tabular form the names of manufacturers and the many instrumental characteristics of seventeen transmission electron microscopes which are in current use. The chart alone is a valuable contribution, for it can serve as the best available guide for systematically classifying the new instrumental features of electron microscopes as they become commercially available. be used for treatment or manipulation of the specimen while it is under observation in the microscope. Some of these devices have recently been described in detail, usually wit,h the aid of schematic drawings. sectional or exploded views. For temperature control, Hedley and McGeagh (187) have designed and constructed a specimen stage enabling temperatures from -150” C. to 2200’ C. to be achieved inside the versatile AEI (;\ssociated Electrical Industries, Ltd.) EM-6. ;\gar and Lucas (220) have discussed a grid heating stage for the ELPI-6. Cooling devices have been described for use with the Siemens (361),the RC.l E11IV-3series (92), and Hitachi HU-11 (467) electron microscopes. For changing the position of the specimen, construction det,ails are available for the rotating specimen holder (1‘74) for the Philips ELI-100, the precision goniometer stage for the Siemens Elmiskop I (270) and RC.\ E1111-3 ( 1 ~ 7 9 niicro) scopes. .\ high-strain specimen apparatus, described by .\ndrews (111, permits strains up to IOOO% to be applied to the specimen during observa-
magnified about lo6 times and at a resolution of 3 A. or better. Lynch (27'4) points out' that the field ion microscope is particularly useful for (359). studying clean surfaces because the high Most microscopists either purchase electrical field forces that are generated their microtomes after making a careful act as a built-in cleaning mechanism. survey of the many excellent' comHe also discusses applications of field mercially available models, or they ion and field electron emission microsmay make modification of t,heir existing microtomes. At lorn cost, Clevenger copy as well as low-energy electron (87) has constructed a microtome which diffraction techniques for studying is based on the principle of thermal changes in surface structure. Brandon specimen advance. Cutting action is (62) has reviewed recent advances in by rod flexion, thereby eliminating the theory and development of the field critical moving parts. His electron ion microscope, citing factors pertaining micrographs of thin sections of biological to image intensification and the admaterial cut with this microt'ome vant,ages of using neon gas. Applicaindicate good cutting action. tions of field ion microscopy were preTwo species of instruments, the sented at' the Kyoto conference on electron mirror microscope and the Metals in 1962. h symposium on field field ion microscope, have become comemission was held at Notre Dame mercially available. I n the General University in 1962. Mills electron mirror microscope, a I n the last biennial review (53) refbeam of electrons advances toward the erence was made to commercially specimen but does not come in contact available microprobes. Recently the with it. The specimen is biased slightly new model Korelco-,\MR/S electron negative with respect to the source of the probe microanalyzer has become incident beam, consequently the elecavailable and can be added to that list. trons in the beam are ~eflectedfrom the This instrument, as do many others, vicinity of the specimen and rearranged combines the techniques of electron in a manner which depends on the elecmicroscopy and x-ray spectroscopy, tromagnetic irregulai-ities associated providing for both qualitative and with the surface of i.he specimen. A quantitative information concerning the magnified image or pattern is produced elemental composition of small volumes which can be observed on a viewing of material. The analyzing goniometer screen and phot'ogra1:hically recorded. can be adjusted to scan an area for the Details of performance, instrumental presence of several elements of interest aims and principles of operation have or it can be set a t a fixed angle for been published (8, 220, 289, 878). indicat'ing variations in concentrations Although the basic features of electron of a given atomic constituent in multimirror optics are not new, Mayer (289), component systems. The new JXh-3 a pioneer in this fiell, indicated the electron probe microanalyzer, distributility of the instrument for observing uted by Fisher Scientific Co., simulstatic as well as dyn2,mic electric and taneously yields six types of data. magnetic domains. ;?or esample, he Some manufacturers of electron studied magnetic domiiin patterns that microscopes, notably Siemens and were induced mechanically in a strained Hitachi, are fabricating x-ray microsilicon-iron sheet (220). Recently, he spectroscopical attachments that can be used the instrument f x the formation coupled with electron microscopes. Alof thin polymer film:; on metal subthough these attachments will further strates by slow electron bombardment extend the versatility of the electron and for the study of the electrical microscope, selection of electron microphotoresponse of these films (290). probe instrumentation will be deterThe photoresponse of 9, thin .\I2Or film mined on the basis of many factors such was also demonstrated (225). as cost, required sensitivity, and freThe portable Cenco field ion microquency of intended application. scope, based on the developments of The symposium on the electron probe Muller, has a demountable specimen x-ray microanalyzer, a t Hargate, holder which permits convenient reEngland, in 1962 has been thoroughly placement of the specimen-emission tip. reviewed by Franks (148). The past, Field ion microscopy is applicable to the present, and future of this kind of study of atomic orgrtnization in the instrumentation was discussed. Nulattice structure of crystals that have merous examples of successful applicamelting points above 1400' C. For tions were cited that contributed to the materials having lower melting points solution of industrial problems and to and for gas absorption studies (16) studies of a more fundamental nature, field electron emission techniques have such as the examination of grain been used. Xccording to hIiiller (817, boundary diffusion in zone-refined chemi$IC?), lattice defects, such as atomcals. .\pplications that may be of vacancies and dislocations, and the interest to the archr.ologist and the art, presence of precipitate clusters can be historian were also mentioned. The use observed as patterns of atomic detail of the analyzer in biological applications tion with the Siemens Elmiskop I. X tilting-stretching stage for applying uniform strain was also mentioned
was just beginning a t that time, and encouraging results were obtained a t the Cavandish Laboratories. Other scientists were also using the microprobe for investigating biological materials. Mellors and Carroll (296) showed that with proper specimen preparation, microtomed sections of tissue were amenable to sensitive chemical analysis by probe techniques. Thermal damage to the specimen was prevented by mounting the specimen on a carbon block and keeping the beam current at about 2 X 10-8 amps. a t a n accelerating potential of 27 kv. They identified and showed the distribution of iron and chromium as a function of position in a section of hip joint capsule taken from the vicinity of a metallic implantation. As a n aid to sampling heterogeneous mat'erials, such as biological solids and petrographic specimens, Wyckoff, Laidly, and Hoffniann (503) utilize preliminary microradiographic and x-ray spectrographic techniques. These techniques are thrn combined with more critical microprobe analysis of selected portions of the gross sample. Yon-conducting, polished sections of bones, teeth, rocks, and minerals are covered with a vacuumdeposited layer of carbon to ensure a steady probe current. Bird (41) has clearly demonstrat,ed the value of electron probe microanalysis in a petroleum laboratory. He gives an excellent account of the versatility of the instrument, manufactured by the Cambridge Instrument Co., and cites good examples of practical applications of sections and surfaces of metals. For example, the stain on the surface of a copper specimen was associated with the presence of trace amounts of sulfur. In another study, factors dealing with the formation, transformation, and dissolution of anodic films on aluminum alloys were discussed by Wood, Marron, and Lambert (501). They reported on the effects of reactions occurring in aqueous media which modified surface porosity and chemical composition of their aluminum test specimens. Clayton (86) reported on the electron probe analysis of aluminum intermetallic compounds a t 10, 15 and 29 kv. After the measured intensity ratios were corrected for absorption, using the correction curves for aluminum published by Castaing in 1960, good agreement was found to exist betwem stoichiometric values and the probe analysis values for aluminum, especially for the 15 kv. and 29 kv. trials. Twentyeight organized morphological units from the Orgueil carbonaceous meteorite were critically examined by electron probe techniques, by Nagy et al. (3%). Their findings are of fundammtal (3011cern because they deal with the 'uspected presence of biological forms w1~ic.h VOL. 36, NO. 5 , APRIL 1964
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are not believed to be due to terrestrial contamination. The qualitative aspects of electron probe x-ray microanalysis have recently been described in two parts. Part I , by Duncumb and Shields ( l S O ) , is concerned with corrections that often have to be applied when the constituent elements vary widely in atomic number, and when absorption and fluorescent effects within the specimen are significant. Their paper emphasizes the correlat,ion of some of the methods proposed for evaluating these effects. In Part 11, .\rchard and Nulvey ( I S ) consider the advantages of using computational methods for evaluating these effects in combination or separately. Birks and Batt (43) have used a multichannel analyzer for the rapid collection and sorting of the various data produced by the electron microprobe. Equipment, operation, and application are discussed in detail. Ignoring fluorescent' enhancement of x-ray emission in effect is considered t (462) outlined a method based on earlier work for correcting the atomic number effect5 in some binary systems of metals. Concentrations, obtained from corrected intensity ratios:,for 23 systems of known composition are listed. Ziebold and Ogilvie (514) review the theoretical and semiempirical methods for relating x-ray spectrometric data to chemical compositions. Their paper includes a listing of measured and corrected values for a number of binary teins. The authors conclude that calculated corrections cannot generally be expected to give the 2% accuracy which is obtainable with carefully prepared calibration samples. The main aspects of the problem of x-ray microanalysis for the light elements are presented in detail by Dolby (127) who considers the use of a proportional counter and pulse analyzer as the best practical approach. Application to the elements Be, C, and 0 demonstrate t,he performance of the instrument. Merritt et al. (502) modified an .\RIA electron probe for detecting carbon by making a pseudocrystal from the successive stacking of about 200 monomolecular layers of barium stearate. The soap film crystal, which was mounted on a 4-inch radius crystal backing plate, had spacings of about 50 A , , thereby overcoming the s ~ ~ a c i nlimitations g of ordinary crystals. The C K CY pulse-height distribution curve from a proportional counter and the scan across the carbon Kcu line illustrated thr potential utility of their method in thepevaluation of carbon-rich materials and other light elements having long Kcu wavelengths. In most electron probe x-ray microanalyzers the produced x-ray beam produced exits a t an oblique angle with respect to the 176 R
ANALYTICAL CHEMISTRY
plane of the specimen. Shirai and Onoguchi ($I 4 ) consider bending the electron beam so that the x-rays could be emitted from the specimen a t a 90" angle, thereby possibly producing more accurate analysis, especially in specimens having rough surfaces. The recent ASTM symposium ( 1 7 ) on x-ray and electron probe analysis has indicated the surge of interest that has developed regarding the instrumentation, the application and the problems surrounding this hybrid appa,ratus. If published, these proceedings will constitute a useful supplement to the earlier ASTM Special Technical Publication, Yo. 317 (18), dealing in part with the electron probe analysis of metals. It appears that the present rate of growth of electron probe technology will justify special categorical treatment in forthcoming reviews of its application to chemical analysis.
TECHNIQUES
Fortunately, many of the new techniques conceived and developed by practicing electron microscopists during the past two years have been generously published in scientific journals or have been presented at scientific meetings (1, 17, 138, 178,220, 375). Communications of this kind have simplified the approach to the solution of many problems and prevented time-consuming duplication of effort. The examination of metals or other crystalline materials in the form of films or foils requires special preparation. Materials may be vacuum-deposited inside the microscope as reported by Pashley and Stowell (220) who, relying on the earlier technique of Bassett (1960), studied the nucleation growth and apparent liquid-like behavior of silver layers. Whapham and Makin (220, 490) transformed uranium foils to KOz by heating the foil on a specimen grid while it was in the microscope. Vacuum depositions may be made outside the microscope and crystal growth induced by various treatment. Thin vacuum deposited films of titanium were oxidized to the rutile form of Ti02 by Cheever, Parker, and Bobalek (80),using stainless steel specimen grids as a support. The intact films, ranging from 150 to 1900 A. in thickness, were examined electron microscopically and by a coordinated electron diffraction method to obtain information about the purity of the deposited titanium layer and subsequent phase transformations on heating. Their techniques and their cited references can be of value where metal and metal oxide are being considered for optical applications. Intact carbon films are sometimes difficult to prepare. Molenaar and
Schotanus (307) describe in detail an improved method for making thin, intact carbon films that lie flat, adhere firmly t o the grid and are quickly prepared. Sometimes a film network is desired for supporting thin tissue sections or as an aid in correcting for lens astigmatism. Byrd (71) outlined a procedure for making polyvinylformal net films which are subsequently coated with a layer of vacuum deposited carbon. The polymer is dissolved in a solvent, leaving a carbon network. Harris (176) produced small holes, 0.05 to 25 microns in diameter, by steaming polyvinylformal films containing glycerol. The glycerol 'content in the polymer solution controls the number and size of the holes. The wettability of copper specimen grids was enhanced by Harris ( 1 7 3 ) who placed a drop of water on the smooth side of the grid and then quickly passed it one or more times through the hottest' part of a Bunsen flame. Thin sections of the specimen were recovered on selected portions of the grid. Hart (176) sublimed tungsten wire below the melting point to produce sharp, relatively grain-free shadows. An electronic circuit for automatic regulation of the temperature of the tungsten wire was presented. The Ladds (255) describe new equipment and techniques for obtaining sharper shadows from evaporated Pt-C pellets, and for handling heat-sensitive and moistlire-sensitive materials. Il'orton and McMullen (335) have used the intense, coherent light of a ruby laser to form holes 5 to 50 microns in diameter in metal discs which are reported to be suitable for electron beam apertures. Measurement of film thickness is often of concern. Estimates can be made from the geometry of the evaporation system and from observations of interference colors of the deposited films (80, 440) ; more critical measurements require application of lightoptical methods, mainly dependent on the use of interference and polarizing microscopes. However, where the film and its substrate are light-opaque, and must remain continuous, other methods must be employed. Ichinokawa and Yamada (219) used the electron probe microanalyzer to measure film thickness as well as to determine the chemical composition of iron-nickel films, over areas of about 1 p 2 , after establishing suitable working curves. Mathews and Buthala (286) overcame the difficulty in stripping vacuumdeposited carbon substrate-films from glass or mica surfaces by a chemical method that eliminates the use of sometimes undesirable parting agents. The method was based on immersing the carbon-coated slide in 25Yc aqueous potassium hydroxide. Squares of carbon film that floated free were recovered
on bronze screens, neutralized in dilute acid and rinsed several times in distilled water. This simple technique should promote the use of carbon substrates which hitherto might k ave been avoided due to difficulty in stripping the carbon or t,o undesirable textures imparted by intervening parting layers. Some crystalline materials have been microtomed into ulvathin sections. For esample, Tice and Lasko (464) have developed a technique for the microtomy of bundles of fine metallic filaments situated in a matrix of aluminum or copper. From the intact thin-sections, the size, continuity, and distribution of the wires could be ascertained. They used selected area diffraction techniques to investigat,e the structure of the metal filaments. Watson (220) studied tactoid-forming colloidal crystals of W03 and p FeOOH in the thin sectioned and unsectioned form. Persson (220) discusses the intricacies of microtomy, emphasizing factors such as speciinen orientation, rate of cutting and reduction of knife chatter, which have to be controlled in order to obtain optimum results. He points out that a fairly small series of high quality sections may give useful information more readily than hundreds of random sections, thus giving rise to so-called rational ultramicrotomy. However, Phillips ( I & ' , 220) cautions about artifacts that ma,y develop due to severe shear deformaticn and the rise in temperature when cry: talline materials are thin sectioned. Stewart and Davidson (439) prepared thin sections from single organic crystals that mere intended for use in abtiorption spectroscopical studies. I t appears that the method might be appli2able to some of the problems encountered in electron microscopy. Their initial attempts to section the crystals produced uncont,rollable fragmentation of the cut thinsection. However, it was observed that the portion of the crystal still mounted in the chuck of the microtome remained intact and appeared to be polished b y the knife action. By facing a single crystal, then reversing the crystal in the chuck and making small advance increments while sectioning, the residual crystal was reduced tc a thickness of 1000 4 . without fragmentation and became the test specimen. For those who want to cut thin sections with glass knives made in their own laboratory, the recommendations for improving knife quality, as described by Fukushi, Brown, and Goode (I&!?), should be useful. Some crystalline mstterials can be cleaved into platelets sufficiently thin for direct observation in the electron microscope or for use as specimen substrates (26, 449). Beer and Highton (30) obtained thin graphite films by the successive fracturing of well formed
graphite crystals near liquid nitrogen temperature. The method avoids the use of adhesives which sometimes are used to recover thin platelets and which may interfere with high resolution microscopy. Preparation of clean surfaces of relatively large crystals by vacuum cleavage has been discussed by Joebstl (227). The vacuum chamber, which makes it possible to cleave, and then to replicate the specimen without breaking the vacuum, is shown in a labelled drawing. Bulky crystalline materials can be thinned by chemical or electropolishing techniques for transmission electron microscopical examinations. .In improved technique for thinning metals has been described by Hugo and Phillips (227) who allowed fine jets of electrolyte to spray vertically onto the top side of the specimens, '/*-inch square or smaller. After thinning, the specimen can be electrolytically cut out of the original specimen foil thereby minimizing deformation. Although not recommended for use with hazardous electrolytes, their method appears to have the advantage of control, speed, versatility, and absence of edge attack. Thinning techniques for many metals and alloys were described recently by Thomas (452). Keown and Pickering (239) describe a chemical thinning technique which they applied to ferrous metals. Their three-stage procedure, which yields specimens about 1000 A. thick, emphasizes the precautions that must be taken during the intermediate thinning stage to ensure satisfactory results. Brittle semiconductor materials have been thinned by a jet chemical polishing method devised by Booker and Stickler (51). Diagrammatic illustrations, a detailed text, and transmission electron micrographs indicate the utility of their method a? applied to fragile specimens of silicon and germanium. Dewey and Lewis (122) describe a polytetrafluoroethylene holder for the rapid preparation of metal foils. The electropolished specimen emerges a$ a disk, thin in the center and thicker a t the edges, which is of a size that permits it to be placed directly in the electron microscope. Bubbles that might form on the specimen during electropolishing are removed by ultrasonic vibrations. Ion etching, neglected for many years, is gaining in popularity as a selective technique for enhancing textural detail in polymers, metals and biological materials. This trend is indicated by Spit (428) who briefly reviews the relatively recent applications of ion etching by others, and then proceeds to describe his investigation of the cellulose fibers, Fortisan and Fiber G, and high and low impact polystyrene samples using gas discharge etching, thin-sectioning and electron
microscopical techniques. Stewart and Boyde (438) elucidated on the structure of mammalian dental tissue. Their ion etching apparatus was designed so that tooth-sections could be bombarded by a n electrostatically fine-focussed beam of ionized argon while the tooth-section was inside the specimen chamber of a scanning electron microscope. Micrographs were produced by the reflected high-energy electrons from a 16 kv. scanning electron beam. X unique feature of this particular technique is that micrographs could be taken while the specimen-surface was undergoing ionic bombardment. The nature of the surface topography of tooth structure, as revealed by this method, indicates that ion etching is a useful adjunct to electron microscopy. Poppa (368) reports on the qualitative results of experiments with a n ion gun inside an electron microscope. Details of the ion gun, the method of specimen mounting, and the experimental techniques are effectively presented. The influence of repeated low energy ion bombardment on the structure of polycrystalline and monocrystalline films, without exposure to the atmosphere, was investigated. He showed that certain cryst,allites with favorable crystallographic orientation in thin films endure the effects of the sputtering process better than do others. I t appears that this method of ion bombardment offers a means for in situ cleaning and thinning of crystalline specimens for transmission studies. Poppa also considered the possibility of micromachining thin silver films by masking them with carbon films and then exposing them to ionic bombardment. Haymann and Gervais (183) described a n ion etching unit designed to operate a t 1 microtorr and a t 1500" C. intended for use in electron diffraction instruments. Turnbull and Ehlers (459) embedded niobium-tin wires in quartz capillary tubes so that the wires could be conveniently polished in transverse section for subsequent light and electron microscopical examination. Special, pre-shaped polyethylene capsules are available (36) for the convenient positioning of specimens that have to be embedded prior to ultrathin sectioning. These capsules, when used with appropriate embedding media, may have application for handling small specimens that may have to be either brittle fractured, etched, ground or polished prior to replication of the specimensurface. Murray (320) describes a replica technique for relocating selected areas of bulk specimens. Many methods of replication have been developed and discussed a t length and it sometimes seems that the possibility of combination of materials and modification of existing techniques have VOL. 36, NO. 5 , APRIL 1964
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been exhausted. However, as new problems arise in connection with the study of the internal organization of consolidated materials, new replica techniques usually follow in the wake. For example, Banerjee, Hauser, and confronted with the Capenos (E?), problem of depicting the relationship between the microstructure of metals and alloys and the detailed topography of their fracture surfaces, developed a modified replica method. They review the commonly used replica methods which, although adequate for revealing the general fracture topography of metals, were found to be unsatisfactory for studying aspects of fracture initiation and propagation. Their method consists primarily of fracturing the metal test bar, etching the fracturesurface, depositing a layer of platinum and one of carbon on the rotating specimen, and finally anodically dissolving the specimen surface. The net result was the production of a replica which combined the main features of the shadowed-surface replica and the precipitate-extraction replica technique. Stickler and Vinckier (440) discuss the various steps required to obtain good quality carbon extraction replicas of fractured m e t d surfaces. T o evaluate proper thick PSS of the deposited layer of carbon, triey place a polished surface of a stainless steel sheet adjacent to the fractured specimen. During carbon deposition the colors of the polished surface go through the sequence: brown, purple, blue and gray. Film thickness corresponding to the blue-gray interference color was found to be optimum, providing both strength and good image contrast, By these methods microscopical features could be related to physical properties of the metal. Because of the high electron-opacity of textile fibers, the fiber microscopist depends on replication, microtomy, or ultrasonic techniques to reveal textural detail. Relying on the earlier replication techniques of others, Sanders (403) has devised a simple modification that minimizes manipulation of the fragile replica films. The fibers are mounted on a sharply curved bow under slight tension and coated with a 200 ,4.layer of vacuum deposited metal and carbon. The fibers are then transferred to a specimen grid, placed in a special gridholder, and the fibers dissolved in a suitable solvent, leaving intact replicas of the fiber surface. hcrylic fibers subsequently dissolved in dimethyl sulfoxide were successfully examined by this method. I n a comprehensive treatment of the role of electron microscopy in textile research, Kassenbeck (232) emphasizes that ultramicrotomy has yielded new information on the internal structure of fibers and has also helped to develop what may be considered an electron microscopical histochemistry. 178 R
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Several useful suggestions were made for the thin sectioning of fibers and for the routine examination of films by other methods. A replication method intended to faithfully duplicate certain cell structures is proposed by Buthala and Mathews (70), who evaporate carbon onto the surface of the cell and then dissolve the cell in alkali. I n their study of cultured rabbit kidney cells, they indicate that this technique, when used in conjunction with osmium fixation and freeze-dry dehydration, minimizes disturbances of cellular constituents and facilitates the study of surface phenomena that might be due to the presence of active agents on the cell surface. Ladd and Ladd (253) showed micrographs of Pt-C preshadowed, carbon replicas of spleen tissue that had been first brittle-fractured, then replicated while held near liquid nitrogen temperature. They also used alkali to remove the spleen fragments from the replica. Bradbury, Rogers, and Filshie (56) applied ultrasonics in an attempt to release cuticle material from the surface of wool fibers without chemical degradation. With welllabelled micrographs it was shown that, despite careful prior laboratory cleaning of virgin wool, the bulk of the ultrasonically treated sample consisted mostly of contaminants such as dust particles, bacteria and keratinized flakes. Only a small fraction of cuticle material was obtained for subsequent chemical study. They recognized that more stringent measures must be taken to clean the fiber surface as well as to improve conditions for ultrasonically obtaining larger amounts of the desired material. The cleaning of wool presents problems, so does the cleaning of glass surfaces (109). Tichane (455) recommends a two-step sodium hydroxide and hydrochloric acid process for cleaning borosilicate glass for high quality applications, such as the formation of uniform film deposits. This light and electron microscopical study, which depends on freshly fractured glass as the cleanest obtainable reference surface, clearly reveals the efficacy of the suggested cleaning procedure. Stilwell and Dove (442) have shown that a line structure developed on soda lime glass exposed to hot detergent solution. The results of preliminary experiments to determine the effect of this anisotropic structure on the morphology of vapor-deposited metal films were discussed. The sampling of particles from noctilucent clouds has been accomplished by Hemenway, Soberman and Witt (290) who depended on the successful firings of a h'ike-Cajun rocket to an altitude of about 7 5 KM. This exposed the surfaces of high purity
substrate to cirruslike clouds which are believed to be composed of meteor-dust and which reflect and scatter sunlight during the short nights of summer. Particles were recovered in the size range 0.05-0.5 micron in diameter with a frequency of occurrence two or three orders of magnitude greater than that associated with rocket flights into noncloud regions. Compositional analysis of some of these cloud particles with a n electron probe yielded evidence of iron and nickel. Electron diffraction patterns have also been obtained although they have not as yet been identified. Analysis is continuing on this interesting aspect of geophysical investigation. Zeitler and Bahr (516, 513) present a photometric procedure which permits weight determinations of electron microscopically visible particles down to lo-'* gram. This quantitative method is dependent on the linear part of the curve that established the relationship of weight per unit area to photometric transmission through the electron microscopical image formed on photographic film. Pertinent details, which include the general principles of the method, schematic optical layout of their integrating photometer, standardization techniques, experimental proofs and the range of validity are presented. Valentine (&I), reporting on the quantitative aspects of electron microscopy of the leprosy bacilli, utilizes a similar method to determine the mean weight of normal and degenerate forms of these bacilli, the weight of their cell walls, and the weight of carbo1 fuchsin stain taken up by the bacilli under various conditions. The ubiquitous latex particles still present problems. One of the problems concerns rapid and accurate evaluation of size distribution of latex particles as viewed in electron micrographs of the dried dispersion. Maclay and Gindler (276) have devised a simple but unique instrument for determining latex particle diameters. They attached the center-adjusting shaft of a 3-inch bow divider to the stem of a pocket watch which was locked in the setting position and the watch spring removed. Thus, the movement of the hands of the watch could only be effected by adjustment of the divider thumb-screw. The separation of the divider points is proportional to the change in position of the hands of the watch. Repeated measurement of latex particle sizes by the same and different operators have indicated the reliability of this device. Other aspects of their paper deal with automatic methods of particle size evaluation and computer data processing. Billings and Silverman (39) with 84 references review the various techniques for sampling the submicron particles in dusts and fumes in ambient urban and industrial atmospheres. Ten different sampling procedures, including the use of sedimenta-
tion, po~ymeric m e m h a n e filters, and thermal and e1ectrostE"tic precipitat'ors, are conveniently listed in table form with other pertinent data and cross references. hpplicaticns and characteristics of membrane filters, which are gaining prominence as a n easy-tohandle substrate for recovering samples of finely divided materials for study by microscopy and autoa,diography, have been described elsewhere (305, 371). The techniques of autoradiography continue to be used in conjunction with the electron microscope to study microstructures particularly in biological materials. Dohlman and Farkashidy ($20) evaluated the different methods of autoradiography used with electron microscopy and Caro (220) described condit,ions which are '2xpected to produce a n autoradiographic resolution of about 0.1 micron. Bachmann and Salpeter (138) have evaluated an experimental emulsion having improved l~ropertiesfor recording the autoradiographic image and they referred to a method for coating specimens which allows the determination of emulsion thickness a t the side of the section. Some general considl2rations of the factors regarding resolving power with tritium autoradiographs were discussed by Hill (197). A high resolution aut'oradiographic-replication method was applied by Dockum (238) to study a met,al specimen containing alpha emitters within a nonradioactive matrix. By evaporating silver onto a suitable substrate a t low pressure (1 X mm. of Hg) and a t low temperature, then sensitizing the siher with bromine under red light, Noyes [337) produced a fine-grained, sensitive emulsion for recording and observing nuclear particle tracks in the electron microscope. This method may have useful application for improving emulsion-specimen contact in specimens not effected by bromine. Kuhn and Harford (243) found that autoradiographs produced by bacteria tagged with iodine - 125, another low energy 6 particle emi.:ter, were comparable to those obtained with tritium. This and other factor:; indicated that iodine - 125 has good potential for use in autoradiographic localization techniques. Smith and Foaizard (421) refer briefly to some applicat: on and development of autoradiography-electron micrography for the localization of isotopically labelled substances and then discuss their method which enabled them to detect the localization of tritium-labelled glucoside in the myocardial cells of frogs and dogs. Their successful results de1:ended on the precipitation of the glucoside within the cells in order to retain it in place during the histological preparation of the tissue. Moyer and Ochs (316) reported on the autoradiography and photomicrography of polystyrene, polypropylene, and
polyethylene to study the microstructures and especially the crystalline forms. As an adjunct to resinography, the potentialities of autoradiography and electron microscopy become apparent. These selected examples of conception, development and application of a variety of techniques, as well as those presented elsewhere (138, 220), attest to the ability of electron microscopists to confront and solve new and challenging problems.
VISIBILITY
As the range of accelerating voltages of electron microscopes increases, there is a greater need to become better acquainted with the factors that provide for visibility. The practical aspects of visibility are dependent on the resolving power inherent in the applied microscopical device, the degree of contrast that results from the interaction of the incident electron beam with the specimen, and the recording of both contrast and image detail on a screen or photographic emulsion. The influence of voltage on theoretical resolving power has been given by Ruska (220). Assuming that the test specimens will provide for sufficient contrast and that instrumental conditions have been optimized, theoretically a resolving power of 3 7 A. could be obtained a t 10 kv., whereas 0.7 A. could be achieved at 1000 kv. The effects of spatial distribution and the mass of atoms in the specimen, as well as the influence of contamination and degradation of the specimen were also considered. De (114) has also reported on the factors that determine optimum resolving power of magnetic lenses. Heide (220) described the causes of contamination and specimen degradation and then elaborated on a means of simultaneously preventing these two undesirable conditions. Using his cold chamber, which was designed SO that a region surrounding the specimen could be cooled to a t least - 130" C. while the specimen remained a t room temperature, good specimen stability was maintained and rates of contamination were from one hundredth to about one thousandth lower than the contamination rate occurring a t room temperature. For those who want to experiment with cold chambers, the simple but less efficient apparatus, previously mentioned ( % I ) , may be a good place to start. Any improvement in lens design that will reduce spherical aberration will eytend the resolving power of electron microscopes. In this regard, the incorporation of an annular slit in lenses, a development ascribed (158) to Wilska, presumably will improve resolving power and useful visibility. especially at low
voltages, beyond the limit mentioned by Ruska. The energy-selecting microscope of Watanabe and Uyeda (220, 477) which makes it possible to examine specimens with monoenergetic electrons of preselected energy value, may also enhance visibility. The viewing screen of the electron microscope may make visible a silhouette image of the object, a pattern associated with the object, or a combination of both. The specimen may be amorphous or crystalline, or both, The optical phenomenon that is perceived may be a well-resolved detail depicting the intrinsic morphology of the specimen or it may be a manifestation of this structure. For example, the moire pattern produced by two overlapping crystalline structures may reveal far more detail than can be observed in the electron micrograph of the separate crystals. We are reminded by Oster and Nishijima (342) t h a t since the moire pattern arises from repetitive crystal structure, dislocations disturbing this periodicity will become visible in the moire pattern. Furthermore, the magnification implicit in the moire pattern makes visible dislocations t h a t are less than the diameter of single atoms. Naiki (326) has relied on the electron microscopical images of a crystal lattice containing a stacking fault and the moire pattern that was subsequently formed by positioning the crystal having the stacking fault on top of a crystal without a stacking fault. Applying the dynamical theory of electron diffraction, the nature of the stacking fault was evaluated on the basis of contrasts in the image. Howie and Whelan continue to make significant contributions to a better understanding of the contrast phenomena associated with microscopical images of crystal defects. Their experimental confirmation of the dynamical theory of dislocation image contrast was reported in considerable detail (213). By way of introduction, they remind us that the visibility of crystal defects in the electron microscope seems to be directly related to altered conditions of Bragg diffraction which result from atomic displacements near the defect, and that a contrast theory should take into account known dynamical diffraction effects. They conclude that most contrast defects observed in dislocations can be explained on the basis of the dynamical theory, providing certain absorption effects are also considered. Absorption and diffraction effects of electron waves observed in images of crystalline material obtained with a 300 kv. electron microscope were reported by Hashimoto et al. (181). Some quantitative aspects of the energy dependence of extinction distance and transmissive power of electron waves were presented. Their study showed VOL. 36,
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that the relativistic correction of the mass of the electron must not be disregarded when considering elastic and inelastic scattering. Burge and Smith (69) reconsider the 1954 Lenz theory of electron scattering, then present new calculations of electron scattering crosssections and a theoretical discussion of image contrast in the electron microscope. Their treatment of this subject represents a redevelopment of the Lenz theory on the basis of newer theoretical descriptions of the atom. The variations of elastic, inelastic and total massscattering and differential atomic scattering cross-sections with the accelerating voltage of the electron beam are thoroughly discussed. The effects of the angular aperture of the objective lens, and the atomic number of the amorphous specimen producing scattering are also mentioned. A shorter paper by these authors (68) recapitulates their earlier work and then considers the factors of contrast and penetration pertaining to thick specimens examined a t high beam accelerating voltages. From their data, they form the opinion that for amorphous specimens the critical mass thickness increases in a favorable manner with increasing beam voltage up to 1000 kv., providing the objective apertures are not too small. Image distortions hinder useful visibility. The correction of distortion in multistage instruments has received precise treatment by Kynaston and Mulvey in a recent paper (251) and general consideration in their earlier work (620). The calculations of Leibman (1952) and Haine (1961) were extended, giving rise to new calculations (261) expressed in terms of the principal dimensions of the instrument and the optical constants of the lenses. I t was shown that by following simple operating procedures, lens distortions can be substantially eliminated. Reisner (220) has experimented with a method for minimizing electrical chargeinduced a3tigmatism in contaminated objective apertures. Using contaminated apertures that produced astigmatism of the order of a few microns, he found it possible to remove the astigmatism by neutralizing the charge with a low-energy auxiliary electron beam. h s usual, a clear description of the problem and details for solving it were presented. Chapman (76) and O’Hara and Beer (540) describe vector methods for correction of astigmatism in instruments that have stigmator or compensation controls. The substitution of sheet film for glass plates in electron microscopy is also favored by Koster and Spiro (244) n h o compared and evaluated commercially available photographic emulsions on glass plates and on cellulose acetate and polyester film bases. It was found that a film manufactured 180 R
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from a polyester base is a practical material for use in electron microscopy. Polyester base film overcomes the undesirable, long pump-down time usually associated with acetate base films and has the advantage of being easier to store and less costly than glass plates. When contrast in the photographic negative is excessive, it can be corrected without loss of image detail by employing the masking technique proposed by Gonzales (162). This procedure is outlined step by step for obtaining suitable masks for negatives on film and on glass. Thus, contrast control can be achieved for many high contrast electron micrographs that otherwise would be difficult to print by ordinary procedures involving manual dodging. The advantage of his unique method is effectively illustrated by micrographs. Fry (1.52)uses a checkerboard grain, which simulates a photographic grain, to demonstrate the basic concepts of the effect of grain on resolving power. He proposes a method for testing the combined effects of grain coarseness, average transmittance, line spacing and contrast on the visibility of sinusoidal test gratings. Ultimate visibility depends on the optimum use of the electron microscope. Fortunately, a major feature of the EhfSX meeting in Denver in 1963 was a symposium on the optimum use of modern microscopes. Hall (158) laid the groundwork with a presentation of the basic principles of electron optics. His remarks on image formation and beam interaction with the specimen were well planned so as to be of value to microscopists of almost any background. Reisner (138) discussed the instrumental factors involved in the use of electron microscopes, decrying the fact that many operators do not utilize the capabilities of their microscopes because their performance checks on the instruments are not sufficiently critical. He outlined valuable, practical methods for maintaining microscopes in optimum condition for highest performance. B e g (158) then spoke on procedures for achieving high resolution, which included consideration of optimum aperture sizes, condenser systems, contamination, focussing and photographic techniques. It was emphasized that the microscopist cannot neglect any of these factors when seeking truly high resolution. The symposium then divided into two sections. In the biological section, Peachey (138) discussed the ever important subject of artifacts in electron microscopy, which were separated into four categories. preparative artifacts, microscope artifacts, selection artifacts and interpretation artifacts. A consideration of all four types is needed for a critical evaluation of electron micrographs. The non-biological section turned to the subject of electron diffraction.
Bigelow (158) gave the major address covering a description of the principles involved, the use of the electron microscope to obtain diffraction patterns, specimen preparation, interpretation of patterns and fields of application. Other contributed papers concerned specific aspects of the general subject, further underlining the need for the practicing electron microscopist to recognize the performance potential of the total instrument. BIOLOGY
The extent to which the electron microscope has contributed to the advancement of biological information is exemplified by Lumsden’s commentary (271) on the collection of fourteen short essays on electron microscopy in the Hritish Medical Bulletin (64) issued by The Medical Department of the British Council. In retrospect, Lumsden reminds us that since the advent of the first crude electron microscopes, about thirty years ago, the somewhat vague concept of the colloidal nature of protoplasm, prevailing a t that time, has been modified so that now cytoplasmic systems can be defined in terms of precisely organized structures. The intricacies of these submicroscopical structures and the unique morphological features which permit their identification are illustrated by the outstanding collection of 26 electron micrographs of representative cells and tissues, recently made available by Porter and Bonneville (570). These micrographs, originating mostly in the Laboratory for Cell Biology a t Harvard University, were taken with a Philips 200 electron microscope and are reproduced as full page images. The authors include micrographs a t low as well as high magnification so that the observer can conveniently correlate the gross aspects of the cell with the fine detail. The clearly labeled micrographs and the accompanying references and comments will surely serve one of their intended purposes which is to supplement and make current the descriptions and illustrations of fine structure of cells and tissue referred to in numerous reference books. As an aid in appraising cell constituents, the contributions of international authorities on cell biology, originating from symposia of the International Society of Cell Biology, are being published in book form starting with the 1961 symposium. The first book, “The Interpretation of Ultrastructure,” R. J. C. Harris, ed., appeared in 1962 (221), and has been favorably reviewed (68, 252). The text is based on a critical evaluation of the validity and the probable biological significance of images observed in electron micrographs. -4 book by
Mercer (300) presents a comprehensive introduction t o the structure and function of cells in a manner which readily enables the techniques of electron microscopy to be fully appreciated as a means of probing cell anatomy and evaluating cell dynamics. Pitelka’s recent book, “Electron-Microscopic Structure of Protozoa,” is considered by its reviewer (394) to bi: a valuable contribution in that it dixusses the cytoplasmic and nuclear structures common to all protozoa; it also emphasizes the diversification of structures and the possible origin of some of the structures t h a t are characteridc of various taxonomic groups. I n cytology the stud11 of fine structure within grosser structure has been advancing rapidly. Entities associated with the cell, such as the plasma membrane, the mil ochondria, the nucleus, the Golgi apparatus, the microsomes, etc., and possibly other units that may have been discovered since this writing, are kecoming areas of specialized investigation. Attempts are being made to compr’3hend reciprocal relationships of all sub-units to one another and to total cell function. For example, the cell meinbrane compels intensive research because of its relation to the highly functional intracellular lamellar systems (386). including mitochondria as discussed by FernandezMoran (145). I n a well referenced paper of fundamental significance, he discusses the limitations of ultrastructural analysis and then proceeds to describe how the tecl- niques of cryofixation, related low temperature preparations, electron TT icroscopy, and diffraction have been applied to study cell-membrane ultrastructure. His work culminates in the correlation of function and strueturf,. Observations of a repetitive structure of mitochondrial cristae were identified with the enzyme complex referred to M the electron transport particle, tl-e existence of which was established b y other techniques. Parsons (348) applied negative staining of thinly spwad cells to the same problem and discerned two types of particles. Sphericd bodies about 80 mp in diameter attached by narrow necks projected in regular arrangement from the surface of cristae. On some outer mitochondrial mtlmbranes hollow cylindrical structures a3peared. Stoeckenius (442) reported similar observations. Sjostrand (418) observed a smaller structural element about 50 mp in diameter in sections of mitochondrial and cytoplasmic membranes fixed by either permanganate treatment or freeze-drying. Syri thesis of these observations into a single structural picture will no doubt be forthcoming with future work. Current emphasis o,i the study of protein synthesis and the genetic code
has directed attention to ribosomes. One result has been the postulation on the basis of physical data of aggregates of ribosomes linked together b y R N A as the functional structure in protein synthesis. Warner, Rich, and Hall (476) purified ribosomes from reticulocytes and by very gentle methods prepared them for electron microscopy. Their work confirmed the existence of the poly-ribosomes and also showed that the individual particles were indeed bound together by a strand of RN-4. Rich (381) has reviewed this development and its implications in the study of protein synthesis. The amount of work being done on the electron microscopy of cytoplasmic organelles and other sub-cellular structures is well exemplified by the large number of papers on such subjects presented a t the Fifth International Congress (220). On the subject of intranuclear fine structure two significant papers are those by Bernhard and Granboulan (34) and by Swift (445) presented a t an American Cancer Society Conference on the Xucleus of the Cancer Cell. The former (34) pointed out that no consistent specific differences between cancerous and normal cells can be shown at the electron microscopic structural level and that with present attainable resolution differences represented by changes in chromosomal structure cannot be seen. However, they pointed out the potential value of cytochemical and autoradiographic techniques in shedding new light on the problem and described some results obtained by existing methods. Swift (445), utilizing formalin fixation, nuclease extraction and uranyl staining methods, localized areas associated with nucleic acids in Ehrlich ascites tumor cells and in cells from the salivary glands of Drosophila. He identified several types of intra-nuclear, RXAcontaining particulates. New sub-microscopical structures are continually being discovered in the course of study of biological materials. For example, the presence of a unique kind of cytoplasmic inclusion associated with mesenchymal cells derived from an excised tumor, classified as a liposarcoma, has been reported by Scarpelli and Greider (405). Their cytochemical and electron microscopical study suggests that a glycolipid-rich material surrounds these 30 mp particles. The sequential changes, involving the formation of empty vacuoles within clusters of these particles, as observed in ultrathin sections, are probably related to an intracellular process of synthesis and secretion. Bacterial cells have been extensively studied by electron microscopy since the first thin sectioning techniques were developed. Chapman (75) has given a brief historical review of this topic
followed by a discussion of the technical problems involved and some of the results obtained. TO preserve bacterial structures and to counteract an inherent susceptibility to explosion damitge, several modifications have been found necessary in the fixation, dehydration and embedding procedures cutomariiy used for animal cells. Structural details of bacteria resulting from the application of such methods by numerous workers were described. I n addition, Chapman pointed out the need for much more work in this area, in particular in the study of morphological changes caused by the action of antibiotics on bacteria. Another excellent review of bacterial structure was given by Glauert (220). Here the emphasis was on describing at high resolution the structural elements of various bacterial species without going into detail on technique. The variability among different bacteria in functional structures was pointed out. Both of these reviews are good sources of references on the electron microscopy of bacteria. A session on bacterial morphology at the Fifth International Congress offered information on many more specific aspects of this field. At the same meeting a separate session was devoted to certain structural elements of a variety of other microorganisms. The cellular structure of some of the higher plants was similarly scheduled as the subject of one group of presentations. In cytochemical studies considerable skill and experimental controls are required to obtain useful electron micrographs which correlate cell morphology with the response of the cell t o specific chemical reagents. Some modification of structure must occur, but the microscopist attempts to reduce unnecessary formation of artifacts t o a minimum. Consequently he attempts to develop new techniques of fixation, staining, and embedding. The preservation of cellular ultrastructure and enzymatic activity was reported in detail by Sabatini, Bensch, and Barrnett (399) who evaluated the effects of different aldehydes, one of which was glutaraldehyde. The reactions for the localization of enzymatic activity are induced after the initial aldehyde-fixation. Subsequent fixation with osmium tetroxide permitted correlation of the final product of the histochemical tests and the fine structure. This significant study, containing 60 references, thereby simplifying certain aspects of this review, is effevtively illustrated with excellent micrographs taken with an RCA EMC-3F electron microscope. Convenient tabulations for the conditions of fisation of rat liver tissue, and for enzymatic activity retained in aldehyde-fixed tissue estimated on the basis of light microscopy are also included. When applying the VOL. 36, NO. 5 , APRIL 1964
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glutaraldehyde fixation technique of Sabatini, Bensch, and Barrnett (399) to mammalian cell cultures, Gordon, Miller, and Bensch (163) observed that certain portions of the ultrastructure were distorted. Adjustment of the osmolality of buffered glutaraldehyde to isotonic levels allowed cells in suspension t o be preserved for electron microscopical studies. They contemplate that presumably cells obtained from blood and other body fluids can probably be studied by this method. Fixative solutions are usually critically buffered for the study of biological materials by electron microscopy. Malhotra (278) suggests that in some instances the role of the buffered fixative may have been over emphasized, perhaps a t the expense of the critical selection of embedding media. He found that when epoxy resins or partially prepolymerized methacrylate were used as embedding media, pancreatic exocrine cells and the convoluted cells of the kidney appeared electron microscopically similar, whether fixation was accomplished in carefully buffered or nonbuffered, slightly alkaline solutions of osmium tetroxide. However, when monomeric methacrylate was used initially, deterioration of parts of the cells, especially the membrane structures, was visible in the micrographs. S e w embedding materials have recently been discussed a t length (138). Regarding fixation, Claude (220) reported on the fixation of tissue in buffered alkaline solutions of osmium tetroxide, compared with fixation in unbuffered slightly acid solutions of osmium tetroxide. Better fixation of the nuclear material and of fibrillar components of the cytoplasm was obtained with the latter solution. A detailed discussion of the technique and results obtained by fixation with osmium tetroxide in distilled water was given in another paper by Claude (86). Davies and Spencer (111 ) studied variations in the structure of frog erythrocyte nuclei which appeared to depend on whether calcium ions were present in the fixative formulation. Grey (168) has presented tu-o charts interrelating specificity, compatability and general properties of common fixing agents. These charts can serve as a useful model for indexing new fixatives as they are introduced for use in electron microscopy. Beer (220) outlines basic categories of staining techniques and Holt and Hicks (205) evaluate some cytochemical staining methods for studying enzyme localization a t sub-cellular levels. Mercer (301) proposes a scheme for section staining, outlining its advantages and limitations. For proteins, Kendall and Barnard (238) describe an electronopaque stain which substantially preserved the fine texture of the test 182 R
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specimen. The method is based on the covalent attachment of thio (-SH) groups to the primary amino (-NH2) groups of proteins in the presence of silver. During the reaction the silver becomes bound, thereby producing electron-opacity. They advise that any staining technique should be applied a t the lowest level of reaction to avoid the introduction of artifacts in delicate biological materials. They also recommend the use of complementary techniques, such as chemical analysis and light microscopy, to provide adequate control. Padgett (345) uses a phosphatebuffered osmic acid fixative and a nonprecipitating lead-tartrate stain for reproducible processing of biological materials which are eventually mounted as thin sections on a durable colloidon methacrylate substrate. Applications of immunofluorescence, which are dependent on the resolving power of the light microscope, have resulted in significant contributions for depicting zones of antigen-antibody reaction a t the cellular level. Sometimes light microscopical immunofluorescence studies are also coupled with the electron microscopy of metalstained specimens, as demonstrated by Wolpert and O’Neill (500), who elucidated the role of the plasma membrane in the locomotion of Amoeba proteus. The streaming motion of protoplasm, as well as other membrane-associated phenomena, are theoretically considered by Kavanau (234) in terms of a metastable protein-lipoprotein-lipid complex manifesting specific substructural differentiations. The extensive bibliography in his paper serves as a convenient review and also accents the many inherent complexities of the problem. The direct labelling of the antibody with an electron-opaque material such as ferritin, instead of with a fluorescent label, has eytended this very useful technique so that antigenic sites can be recognized at the subcellular level with the electron microscope. The technical applications, the problems, and a partial review of the literature of ferritin labelling for immunology with the electron microscope are presented by Metzger and Smith (303). Among the problems they refer to are those of the selection of the least deleterious fixatives and the development of more specific immune agents and more sensitive labelling techniques. Regarding the latter, Sri Ram, et al. (431) have reported improvements for chemically conjugating ferritin to the protein antibody. Instead of depending on diisocyanate to complex the ferritin t o the antibody serum, they used the reagent difluoro-dinitrodiphenyl sulfone (FNPS) which has the advantages of being milder and stoichiometric in reaction with the anti-
body. Thus, there is little or no loss in the precipitating capacity of the antibody as was the case with the diisocyanates, which sometimes appeared t o polymerize on the surface of the antibody, producing unpredictable yields of ferritin-antibody conjugate. Relying on the ferritin-conjugated antibody method, Mott (314) studied the localization and transformation of the immobilization antigens of Paramecium aurelia. Ferritin granules, conjugated with antibody specific for this antigen, were found on the pellicle and the cilia of these single-cell animals. The application of a counter-stain, consisting of potassium permanganate and uranyl acetate, further enhanced visibility. The selection of the proper embedding agents is a requisite for preserving structure in ultrathin sections of biological materials and in industrial materials such as minerals (502) and synthetic fibers (98). A symposium on the embedding of cells and tissues a t the ERISA Meeting in Denver (138)yielded useful information on old and new formulations. Luft (138) reviewed the requirements that should be fulfilled by embedding materials. These concerned ease in infiltrating the tissue, avoidance of damage to tissue during hardening, ease of cutting, and stability in the electron beam. Some of the undesirable features of methacrylate embedding, also observed by others (278)j were overcome by Watson (138), who discussed two methods for minimizing the objectionable effects of the monomer as it polymerizes. Schreil (138) described a polyphase polymeric material that on final polymerization produces a high concentration of crosslinks. Leduc (158) recommended embedding in a water-soluble glycol methacrylate when cytochemical techniques are t o be carried out directly in the thin section. With this system, dehydration of the tissue can be accomplished in the monomer, one of a variety of conditions of polymerization of the monomer can be selected, and the final ultrathin sections are more amenable to penetration by certain aqueous enzymes than are those embedded in other media. Deaver (138) made solubility studies of reagent and materials used in embedments for ultramicrotomy. Spurr (138) referring t o the work of Staubli (434) and Freeman and Spurlock (149), reported on progress in using epoxy-resin blends as embedding media. The water soluble glycol methacrylate and some of the epoxy resin blends are commercially available in kit form (367) along with well-referenced data sheets describing physical characteristics and the recommended formulations for specific applications. The foregoing references indicate
progress during the pas, two years in the electron microscopy of cells. It is significant that some of the problems that existed in 1962 were partially solved in 1963, particularly with respect to the development of more amenable aldehyde fixatives, 1111x8 reliable ferritin-antibody conjugates, and electronopaque selective stains. Accomplishments of this kind reflect the ingenuity of scientists investigaiing the minute confines of intr:tcellular space. Virus research continues to be one of the most activi? and fruitful fields for electron microsi2opy. The Fifth International Congress for Electron Microscopy held in Philadelphia in 1962 (220) included five sessions on virology and the 1963 meeting of the Electron Microscope Society of America held in Denver (138) devoted an afternoon t o that subject. A dramatic explosion in knowledge of virus fine structure was triggered by the technique of negative staining (63). Horne and Wildy (807) reviewed the use of this technique in virus structure studies and Bradley (61) contributed an investigation of the optimum conditions and materials for the method. The use of negative staining in conjunction with other techniques has also proved valuable. Almeida and Howatson (4) were akle t o determine the fine structure of moi~phologicallyunstable viruses by cutting frozen sections of infected cells and following with negative staining. Humnieler, Anderson, and Brown (218) identified negatively stained poliovirus particles of different antigenicities by means of electron microscopic observations of their agglutination with specific antibodies. Bayer (220) used a similar technique with vaccinia virus. Chatterjee and Sarker (280) combined uranyl acetate treatment with negati {e staining and made the interesting observation that 5 M uranyl acetate removed t h e D N A core of vaccinia. Smith and Melnick (423) used the combination of negative staining with phosphotungstate, positive staining with uranyl acetate and treatment with nucleases t o classify viral nucleic acid as D N A or RNA. The application of negati {e staining to particle counting minimizes the possibility of counting non-viral particles and permits differentiation between full and empty viruses. This method was used by Watson, Russell, and Wildy (484) for herpes virus and Barrera-Oro, Smith and Melnick (24) for human wart virus. Other new methodology for particle counting was reported by Pinteric and Taylor (364) and Smiih and Melnick (422). The ferritin-labelled antibody technique in virus research was reviewed by Morgan e2 al. (91) with specific reference t o influenza and vaccinia. Markham, Frey, and Hills
(883) developed novel optical methods for analyzing electron micrographs of virus fine structure by which the amount of information derived therefrom is increased. Although the development of an all inclusive scheme for classifying viruses remains a problem, its solution has been advanced by data on virus fine structure obtained by high resolution electron microscopy. Wildy (491) discussed this in a review article on virus classification based on symmetry and structure. Andrewes (10) made fine structure one criterion in a treatment on the classification of viruses of vertebrates, while Lwoff, Horne and Tournier (91) proposed a n overall system of classification including both plant and animal virus fine structure. Electron microscopy contributed to the thorough presentation by Casper and Klug (91) of the physical and mathematical principles involved in the construction of regular viruses. Three major structural groups of viruses have thus far emerged-those with cubic, helical and complex or combined symmetry. The first group most commonly have 5 :3 :2 icosahedral symmetry with specified numbers of capsomeres in the polyhedral shell, depending on the size of the virus. The newly classified picornaviruses (299) are 15 t o 30 mp in diameter and apparently icosahedral. Mayor (138) believes a rhombic triacontahedron t o be a better geometric model for this group. Melnick (298) has proposed the name papova for another sub-classification of icosahedral viruses which have 42 capsomeres and a diameter of about 45 mp as represented by papilloma, polyoma and simian vacuolating agent SV 40. Parsons (349) found that the K virus of mice belongs t o such a class on the basis of size and surface structure. Mattern (287) and Mattern, Allison, and Rowe (288) and Caspar and Klug (91) questioned the structure of the papova viruses and suggested a size of about 50 mF and a surface of 92 or possible 72 capsomeres. Mayor and Melnick (292) defended the 42 capsomere structure and Bernhard, Vasquez, and Tournier (35) arrived a t the same figure in a study of the SV 40 virus. More recently Howatson and Crawford (210) disrupted polyoma and papilloma virus particles counted complete sets of capsomeres and again obtained a figure of 42 for both viruses. Crawford and Crawford (99) have since esamined negative stained preparations of polyoma virus mixed with four different strains of papilloma and showed a consistent size difference between the two which correlated with sedimentation data. Inclusion of both viruses in the papova group depends on the final definition of criteria. The larger the virus, the easier its
structural interpretation. Thus three different laboratories (160, 228, 466) have reported similar data on the fine structure of reoviruses, to wit, that they are icosahedral with 92 capsomeres and 60 t o 70 mp in diameter. Polson and Deaks (366) tentatively identified African horsesickness virus with this structural group. The wound tumor virus of plants (220)has also been shown t o have 92 capsomeres in a n icosahedral arrangement. Herpes type viruses have 162 capsomeres arranged in an icosahedron which is enveloped in another membrane. They are sometimes seen in the naked form without the outer envelope. Cruickshank, Berry, and Hay (101) put laryngotracheitis virus in the herpes category and Watrach (482) found intranuclear filaments associated with this infection. Lutzner (273) described the fine structure of zoster virus in human skin as similar t o that of other herpes viruses. Plummer and Waterson (365) identified similar viruses associated with equine herpes. Still larger are the adenoviruses all of which have been found t o possess 252 capsomeres in a n icosahedral capsid. The capsomere count of GA4Lvirus, the adenovirus of chickens, is now firmly established as 252 in agreement with others of this group (483). The CELO virus of chickens has been shown t o be an adenolike agent resembling GAL in structure and mode of replication (355). Archetti and Steve-Bocciarelli (14) identified several simian agents as adenoviruses based on fine structure determined by negative staining, In further work on human hepatitis Truffelli et al. (138) indicated that the previously identified ribosome-associated 18 t o 22 mp particles coalesced into a 40 to 50 mp icosahedral arrangement but no attempt was made t o classify the agent on this basis. Although rod-shaped viruses with helical symmetry such as tobacco mosaic virus are quite readily studied by electron microscopy, the fine structure of animal viruses with helical symmetry is more difficult t o analyze since the helices are usually coiled within a n envelope. Thus Hart (177), using tungsten shadowing, resolved surface structure periodicity of tobacco mosaic virus which agreed well with the 23 A. helical spacing found by s-ray diffraction. -4 somewhat similar helis with different spacing and shape of subunits was resolved by Russell and Bell (397) for beet yellows virus. In the realm of the animal virus Waterson (479) pointed out structural differences between the helices of two groups of mysoviruses represented by influenza and Newcastle disease virus, but within the influenza group reported no significant structural variation among the A , B and C strains of influenza or between human and animal strains of Type A (481). VOL. 36, NO. 5 , APRIL 1964
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Cruickshank et al. (102) and Norrby et al. (834) classified canine distemper virus as a myxovirus on the basis of structure. The third class of viruses with complex symmetry includes the large pox viruses, bacteriophages and others. Vaccinia (338), rabbit pox (489), ORF (323), and bovine papular stomatitis (324) have all been shown by negative staining of intact particles to have surface structure consisting of long winding fibrils sometimes with a beaded appearance. Friedman-Kien and coworkers photographed a similar pox virus isolated from a human case of milker's nodules (151). By varying pH and medium Mendelson et al. (138) unwound the surface fibrils of molluscum contagiosum virus and also found long beaded strands. By the use of negative staining a t p H 10.5 Peters and Muller (356) observed a triple tubular structure of the D N h containing core of vaccinia particles. Howatson and Whitmore (211) and Mussgay and Weibel (321) described the cytoplasmic development of vesicular stomatitis virus and its bullet shape. By means of new preparative and cytochemical techniques electron microscopy is contributing valuable information relevant to the basic problem of virus-cell interaction, with much emphasis on the relationship of viruses t o tumors. Haguenau and Hollmann (171) contributed a valuable study of the problem of recognizing in sectioned tumor cells non-viral particles which resemble known viruses. Bernhard and Tournier (91) pointed out the potential value of combined cytochemical methods in the electron microscopic study of virus infections. Epstein (220) suggested that the presence or absence of a membrane around virus particles can be a valuable indicator of certain structural and functional properties. The effect of saponin and digitonin on biological membranes was utilized by Dourmashkin, Dougherty, and Harris (128, 129) to reveal a hexagonal array of pits in the surface of Rous sarcoma viruses and that of the cell membrane from which they emerged. The presence of adenosine triphosphatase activity in viral membranes was demonstrated by Novikoff, de Th6, and Beard (336) and de Th6 et al. (120) in the case of myeloblastosis and by Epstein and Holt (139) in the case of herpes. In both cases the enzyme activity was also present in the host cell membrane. Watson and Wildy (485)showed by electron microscopy that antiherpes serum agglutinated naked viruses while anti-host cell serum preferentially agglutinated enveloped virus. Relevant to this also Morgan, Hsu, and Rose (312) demonstrated with ferritin-labelled anti184 R
ANALYTICAL CHEMISTRY
body that the empty sacs of incomplete influenza virus could be identified immunologically with fragments of cell membrane. Berkaloff and Colobert (33) by staining sections of infected bovine cells with alcoholic phosphotungstate were able to visualize the surface spicules of influenza virus and t o show by means of hemagglutination and hemadsorption experiments that identical spicules were produced on the membranes of infected cells. Studies of these kinds offer the possibility of adding cytochemical data to the description of virus surface structure and suggest that while the host cell membrane is the source of some viral membranes it probably undergoes infectioninduced transformation before incorporation into the virus particles. Viral nucleic acid extracted from intact particles has been subjected to electron microscopy with good results. Kleinschmidt, Burton, and Sinsheimer (242) mere able to isolate and photograph DNA from $X174 bacteriophage in its replicative form. The D N A was cyclic as in the non-replicative form but was a double helix rather than a single strand. Weil and Vinograd (487) in similar experiments with polyoma viral DS.4 also demonstrated cyclic helix and coil forms. Sharp and Galasso (220) developed a special centrifugation method to insure rapid adsorption of virus to host cells in time studies of infection. Mathews and Buthala (220) took advantage of this method and obtained pictures showing successive appearance at, and emergence from the cell membrane by newly formed vaccinia particles. Dales (104) and also Sielsen and Peters (331) photographed vaccinia being phagocytized by host cells, the former also including for comparison adeno 7 virus which was found intact close to the nuclear membrane. Holmes and Watson (204) showed that enveloped herpes viruses adsorb to host cells more readily than naked viruses and after penetration virus particles were found in pinocytotic vesicles. Hinz, Barski, and Bernhard (198) pictured early nuclear changes and cytoplasmic virus development of encephalomyocarditis in embryonic mouse cells i n vitro. Dales and Franklin (107) studied the same virus in L cells and compared it with mengo virus. ;2lmeida, Howatson and Williams (6) described virus maturation in nucleolarassociated aggregates in human warts. Dales and Choppin (106) with influenza and Mussgay and Weibel (322) with Newcastle disease virus demonstrated that the intact particles were phagocytized by the host cell and then lost their identity. Hoyle, Horne, and Waterson (214) isolated from normal chorioallantoic membrane of fertile eggs particles which were agglutinated by
influenza virus and may be involved in the first stage of influenza infection in this host. Moore et al. (308) combined negative staining and shadowing to show the difference between complete and incomplete influenza particles, Barry, Waterson, and Horne (25) demonstrated that multiplicity reactivation of ultraviolet irradiated influenza produced a pleomorphic virus population similar t o that obtained by the Von hlagnus technique. Blough (47j 48) showed that pretreatment of fertile eggs with vitamin d alcohol, certain detergents or lipoxidase altered the allantoic membrane cell surface so as to favor production of filamentous influenza viruses thus indicating that the nucleoprotein helix conforms to rather than determines the shape of the virus particle. 13y negative staining of whole mounts Choppin (83)obtained excellent pictures of filamentous influenza viruses emerging from monkey kidney cells i n vitro. Bayer (138) had evidence that the mucoprotein hemagglutination inhibitor of influenza has periodic structure which matches the surface projections of the virus. Schafer (406) used electron microscopy in studying the significance of the separate components of the Maus Elberfeld, fowl plague and Newcastle disease viruses. I n addition to observing the biological activities of the individual components he combined isotopic labelling, fluorescent antibody methods and electron microscopy to trace them separately in cells originally infected with intact viruses. The para-influenza viruses have been shown by electron microscopy to be myxoviruses of the Sewcastle class by Waterson and Hurrell (480)and also by Hermodsson and Westman (194). Bukrinskaya (66) using autoradiography and electron microscopy indicated that the protein component of Sendai virus remained outside the host cell membrane in contrast to other observations that myxoviruses are phagocytized in toto. The common use of cultures of monkey tissues as in vitro hosts for viruses in research and vaccine production lent emphasis to studies of simian viruses. Gaylord and Hsiung (156) first reported the morphology and intranuclear replication site of the simian vacuolating agent SV 40. Subsequently Mayor and co-workers have used electron microscopy in biophysical studies (291) of the virus particle and cytological studies (293) of its growth cycle in monkey kidney cell cultures. Granboulan et al. (166) have also done detailed studies of this type. Simian viruses morphologically similar to human measles were described by Lokhova and Voronina (268) and by Stefanov (435). In monkey kidney cells Granboulan and Wicker (167)
established that virus W , a latent virus of baboons, also has a structure typical of myxovirvses. Cellular langes due t o infection with reovirus R c’re reported by Rhim, Jordan, and Mayor (579) for t j pe 1 virus and by Gomrtos et al. (160) for type 3 virus. The viruses were synthesized in the cytoplasm and formed matrices of completed particles. Da1t.s (105) observed the association of recwiruses with the host cell’s spindle apparatus. Rabies virus came under the scrutiny of the electron beam in 1962 and 1963. Matsumoto (286) and Roots (592) both found cytoplasmic round and elongated particle> in sections of rabies-infected mouse brain. hlmeica et al. ( 5 ) and Pinteric, Fenje, and Almeida (363) found myxoviruses associated with rabies infection in tissue cultures and in mouse brain respectively. iltanasiu and co+orker? (20) and Davies et al. (112) using different host-virus systems showed round and elongated particles both inside and outside the cell with budding a t the cell ml.mbranes. Some of the extra-cellular particles appeared t o be myxoviruses. Srhoen, Shumkina, and Vanag (407)and Sokolov and Vanag (425) combined elec tron microscopy with immunofluorescence to identify a rabies antigen in Negri bodies, implying that this is the site of .tt least one stage of virus synthesis. Lipovirus, an agent isolated from an infectious hepatitis pat lent, was studied by Dunnebacke (13:) in liver cell cultures. Aggregates of ring-shaped bodies appeared as the product of infection. Aggregation of some viruses may be caused by interconnecting DNA strands according to findings of Smith and Wallis (138) with herpes virus. Almeida, Cinader, and Howatson with wart and polyoma viruses ( S ) , as well as Lafferty and Oertelis (264) and Bayer and Mannwiler (28) with influenza virus photographed negatively stained virus-antibody system in which the rod shaped antibody molecules could be recognized attached t o virus particles and a t optimum concmtrations linking them in aggregates. The subject of viru,-induced tumors has been reviewed in depth in the first volume of a new series of monographs on “Cltrastructure in Biological Systems” (,$SO), and the electron microscopy of tumors and tumor viruses is included in other cancer reviews (72, 84). The structure of tumor viruses has been discussed by Howatson (bog), Dmochowski (124) arid others. I t is apparent that morphologically they represent a a i d e variety of viruses. Lyons and lloore (275) purified and examined by negatiiie staining the mammary tumor agent of mice and found myxovirus-like particles. Feldman (143) thoroughly studied by thin sectioning the origin and distribution of
viruses associated with mouse mammary tumors. Miyawaki and Nishizuka (306) observed precancerous lesions and associated viruses in mammary glands of three strains of inbred mice. The avian myeloblastosis virus was reported by Eckert, Rott, and Schafer (136) t o be a myxovirus. Bonar et al. (49) confirmed this and found similar structure for erythroblastosis virus. The cell response in the thymus of myeloblastosis-infected chickens was studied by electron microscopy by de ThB and coworkers (121) while Heine et al. (188) found the virus also in the pancreas. The polyoma virus, an icosahedral oncogenic particle was separated by Crawford, Crawford, and Watson (100) and also by Winocour (496) into an intact fully infectious form of different density from an empty non-infectious type. Rous sarcoma virus fits structurally into the myxovirus classification and its intracellular elaboration was observed by Heine et al. (189). Vogt and Luykx (468) recorded changes in cell surface structure associated with Rous infection. Specific sugar-induced changes in mitochondria, nuclei and endoplasmic reticulum of Rous sarcoma cell. were described by Voelz and Levine (138). Parsons (547) by the use of the cell spreading technique showed that the Gross leukemia virus resembles the Rous sarcoma agent in having an envelope covered with fine projections. The fringe, however, differed from that of the mouse mammary tumor virus and it was suggested that such differences might be applied in classification schemes. The report by Dalton, Haguenan, and hfoloney (108) of tailed viruses associated with the hloloney leukemia of mice was the first correlation of such structures with a specific animal virus disease. Zeigel and Rauscher (511) more recently made a similar o b e r v a tion in the case of Rauscher leukemia and Rich and Johns (586)with Friend leukemia. De Harven (158) urged caution in identifying tailed animal viruses in view of the existence of similar structures of nonviral origin. Zeigel and Rauscher (138) showed that physico-chemical stresses have much to do with the preservation of tails and other morphological features and related biological activity, emphasizing the importance of preparative techniques in the electron microscopy of viruses. Granboulan and Riviere (165) found viral particles in 21 of 37 spontaneous AKIl leukemias particularly in the thymus and in 4 of 7 spontaneous lymphomas of mice. De Harven (158) discovered in normal mouse thymus particles identical to mouse leukemia viruses and discussed their possible
significance in the role of the thymus In leukemogenesis. Myxoma viruses in electron microscope studies by Padgett et al. (3.44) and Chapple and Restwood (77) were seen to have the structure of pox viruses. A transmissible agent which elevates plasma lactic dehydrogenase levels of mice was isolated by Riley (536) from mouse tumors. Bladen and Notkins (44) found the activity to be associated with a pleomorphic particle which by electron microscopy was 70 t o 7 5 mp in size possibly with a tail structure. Efforts to identify by electron microscopy virus particles related to human tumors have had some apparent success. Lunger, Lucas, and Shipkey (272) extracted particles from the milk of normal and breast cancer patients and found significant differences in both quality and quantity. Some of the tumor-associated particles resembled those from mouse mammary tumors. Zucker-Franklin (518) photographed virus-like particles found in the lymphocytes of a patient with chronic lymphocytic leukemia and Shubin (415) observed 40 t o 50 mp particles in blood cells of hemocytoblastosis patienti as well as in tumors of the parotid gland of man. The Yaba virus, associated with histocytic tumors of man and monkey, was investigated by Owens, Jletzgar, and Grace (545). They found typical brick-shaped pox viruses in the tumor cells. These lines of investigation \\ill undoubtedly continue to be pressed vigorously by electron microscopists. K a y (448) in a review of the morphology of bacteriophages pointed out the great variety in their functional structures especially as regards the manner of infection. The fine structure of the infectious 114s and non-infectious 70s particles of bacteriophage dX174 was analyzed by Daems et al. (103). By negative staining both appeared to be icosahedrons of the same size with twelve sub-units. Different intensity of uranyl acetate staining indicated that the relative weights of the particles resulted from difference in size of the single strand of DNA within. Stouthamer, Daerns, and Eigner (444)showed that both types of particles were adsorbed to host cells and gave up their DXh. The mechanism of infection by tailed T6 phage na. compared in the same report CotaRobles and Coffman (g4) made a detailed study of the interaction of T 2 coliphage with host cell malls and showed localized cell wall degeneration and withdrawal of the virus ghosts after infection. Crystalline aggregates were found by Schuartz and Zinder in E. coli infected with the small f2 phage (410). Marvin and Hoffmann-Rerhng (284) identified and characterized two new small bacteriophages. Eiserling and Romig (137) studied the SP3 phage VOL. 36, NO. 5 , APRIL 1964
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of B. subtilis and found differences in structure as compared with coliphages. Davison (113) resolved icosahedral fine structure in the head of the B. subtilis phage SP8 as well as structural changes associated with triggering the tail. Smith (448) discussed the unique features of insect viruses including their tendency to be occluded in capsules or protein crystals within the host tissues. The development of the tipula iridescent virus particle was investigated in a time study by Bird (40). Bergold (32) examined the molecular structures of some insect virus inclusion bodies, and Grace (164) showed that the virus of cytoplasmic polyhedrosis survived in insect cells grown in vitro. Steinhaus and Leutenegger (436) identified an icosahedral virus in a scarab, the first such found in a coleopterous insect. Recent high resolution instrumentation and techniques have made possible electron microscopical investigation of structure in the size range of proteins. Levin has applied negative staining t o the revelation of the structure of several protein molecules. Cytochrome C (261) observed in various states of denaturation appeared as a long rod folded in three sections into a compact structure 40 A. long. The four structural units of hemoglobin (264) were oval rings about 40 x 50 A. each of which resembled accompanying pictures of myoglobin molecules. Erythrocruorins of lumbricus (263) were complex molecules 260 X 160 A. consisting of twelve elements arranged in two contiguous six-sided rings. A still smaller structural subunit was also resolved. The molecule of 248 hemocyanins from Crustacea (262) were about 100 X 200 A. and made up of two large subunits, an observation which Levin translated into a model consisting of two triangular sections with axes at 90'. Van Bruggen, Schuiten, and Wiebenga (463) obtained micrographs of the 168 and 238 components of Crustacea hemocyanins closely resembling those of Levin but interpreted them t o show a molecular model of the 16s component consisting of eight subunits situated at the corners of a cube with the 23s component being a dimer of this structure. In the same paper were shown micrographs of hemocyanin molecules from three Gastropoda suggesting in two cases a molecule composed of six equidistant parallel layers of subunits and in the other three double-layers. The difference correlated with sedimentation studies of dissociated molecules. Zobel and Carlson (616) found the molecule of myosin to be a rod-like structure with a globular head, and considered the relation of aggregates of them with the A-band filaments of muscle. Horne and Grenville (206) dissociated L-glutamate dehydrogenase into its major subunits and resolved structure at the 186 R
ANALYTICAL CHEMISTRY
next level of organization. Each subunit was described as a group of boomerang-shaped structures forming a tetrahedron. POLYMERS
The persistent striving t o devise and apply electron microscopical techniques and instrumentation to polymers and resins has resulted in a better understanding of their structure and behavior. Many papers representing endeavors in the field have been published, including symposia ( I , 19). They encompass attempts t o define further the sizes and shapes of isolated molecular, micellar, and crystalline units, to resolve the constituents of heterophase polymeric materials, and to offer explanations for the mechanism of certain reactions. For example, Wishman et al. (498) combined electron microscopy with ultraviolet microscopy to study the influence of methyl-vinyl pyridine and total monomer content on the sizes, shapes and chemical constitutions of particles formed in the early stages of batch polymerization of acrylonitrile/vinyl acetate/2-methyl-5vinyl pyridine with a chlorate-sulfite redox catalyst. The copolymerization of acrylonitrile with sodium p-styrenesulfonate in aqueous solutions was investigated by the team of Izumi, Kiuchi, and Watanabe (223). Their work on the effect of initiator (ammonium persulfate) concentration on the copolymerization rate was beneficially aided by direct observation with the electron microscope. This study showed that sharp rises in copolymerization rates within 0.2 t o 5 mole % could be reasonably interpreted in terms of the emulsion copolymerization mechanism. Bradford and Vanderhoff (1, 59, 60) review the importance of electron microscopy in research and development of synthetic latexes. This is a broad, interdisciplinary field that involves particles as small as 20 A. and as large as 10 microns. Since the electron microscope can be used over the entire range of sizes and also in studies of size and shape distributions it is the primary tool. The authors review their techniques, including bromination to harden the globules and preserve their sizes and shapes. The authors also studied the mechanism of film formation and the morphology of resultant films with and without pigment. Rupar and Mitchell (396) examined the morphology of synthetic latexes from the swollen monomer-polymer particles a t low conversion to the final latex particles after removal of the unreacted monomer. -41~0,they characterized the effect of different polymerization treatments on the size and size distribution of the latex particles.
Certain styrene/butadiene/methacrylic acid latexes form discontinuous films on drying (169). Guziak and Maclay (169) found that these films can be peptized to form a reconstituted latex containing spherical particles of the same size as the original latex particles. The property of redispersibility of transparent flakes of polymer in water is attributed to limited particle coalescence in the granular polymer. The incorporation of high numbers of carboxyl groups in the polymer is essential for redispersions. Ionization of the carboxyl groups on the particle surfaces upon addition of a base develops repulsive coulombic forces causing particle separation. The inability to redisperse the film a second time is thought to be due to polymer plasticization or to the migration of carboxyl groups to the particle interior as a result of coalescence forces. The electron microscope has been used to study the surface textures of dried and aged paint films (339, 517). The study of the polymerization of vinyl acetate in aqueous media was performed by Wapper (327), Yapper and Alexander (328). I n the first part (327) of the investigation, the kinetic behavior of the system was observed in the absence of stabilizers. I n this system, polymerization occurred primarily within the polymer particles which formed a stable suspension. Part I1 (528) is concerned with the effects of the addition of a neutral, anionic or cationic soap on the heterogeneous polymerization of vinyl acetate in aqueous solution. The results were interpreted in terms of the absorption of the soap onto the surface of small polymer particles, and the resultant reduction or promotion of the coalescence of these small particles. With respect to cellulose polymers, the gel content is one of the more important factors to control in the production of viscose. Sperling (427) characterizes the small gels present in cotton linter viscose, prehydrolyzed sulfate wood pulp viscose and sulfite wood pulp viscose by light scattering, chemical methods and electron microscopy. It is speculated that the gels from cotton linters and wood pulps, which bear a striking resemblance to each other, may have a common origin. With a different approach, Hermans (192, 193) has studied the flow properties of gels of microcrystalline cellulose and the effects of added electrolytes. Electron microscopy proved a valuable aid in obtaining pertinent data concerning the geometry of the microcrystalline gel particles, an important parameter in fundamental consideration of gel structures and their flow properties. I n the promising field of solid state polymerization Morawetz ($IO), using
carbon replicas, explores the spontaneous polymerization of vinyl monomers on the surface of p-benzamidostyrene crystals. Nucleation occurs in rows which probably correspond to a crystallographic direction. David et al. (110) investigated .;he solid, state polymerization of P-propiolactone initiated by x-ray sources. Rate of polymerization and molecular weight have been measured and the ordered fiber-like structure of the polymer was indicated by x.ray diffraction, spectroscopic and electron microscopical techniques. I n a n arrest>ingpaper, Mayer (290) follows the formation of thin polymer films on metal substrates bombarded by slow electrons in an electron mirror micrcscope. The dependence of growth rate on voltage and the aspects of electrical photoresponse were discussed. Electron microscopy often provides sufficient background information about the morphology of polymeric systems to facilitate 1;he proper interpretations of patternis which are obtained by other means. This is amply demonstrated by the reliance of Rhodes and Stein on electron micrographs as an aid in interpreting patterns produced by light scattering (580). Richardson (583, 584) developed the spray techniques of Boyer and Heidenreich (1945) and of Sicgel Johnson and Mark (1950) for isolating macro. molecules by determining the optimum proportions of good and poor solvents of proper relative volatility to obtain and preserve spheres of large macromolecules (500,000 and greater in molecular weight). H e estimates his accuracy to be better than 20% around one million molecular weight anc! better still a t three million. Peck rind Moore (365) have continued their s h d y of relatively large particles in solutions of polyethylene after concentrating them somewhat by filt'ration. The authors cast and calendered films from the concentrated solutions and revealed t'he particles by etching or stretching. As a result of this new wwrk, the authors confirmed their previous conclusion that they had observed single, extra large macromolecules cif branched polyethylene. I n a paper concerning the size, shape and flexibility of the rubber molecule, Schulz and Mula (409') determined the molecular weight distribution in natural rubber. Walters (474) in clever experiments studied replicas of rupture surfaces of elastomers. The replicas were made while the adjacent untorn part of a specimen was still under stress. He found a strength-producing structure composed of fibrils or:iented along the axis of extension. The structure was basically the same whether or not the elastomer contained carbon black. Medalia, Hagopian ttnd Hall (294)
studied the emulsion copolymerization of butadiene and styrene in the presence of carbon black. I n general, they found that carbon blacks inhibit and retard free radical polymerizations in bulk and solution. After establishing conditions for reasonable rates of polymerization, stable latices were formed that showed evidence of association between some of the carbon black and some of the latex particles. The coagulated rubber stocks, when compounded and cured, gave properties similar to those of dry mixed stocks. The nonohmic behavior of carbon black-loaded natural rubber vulcanizates was studied by Van Beek and Van Pul (462). By comparing systems having good, moderate and poor carbon black dispersions, they discovered that the degree of dispersion could be correlated with field emission characteristics. Using replica techniques to study the fractured surfaces of carbon black master-batches, Prestridge (373) has shown that dispersion, grinding and bonding were the most important factors for evaluating the quality of the black. By applying these criteria, the performance of a tread stock made with such master-batches can be reliably predicted. The electron microscopists were able to study the dispersion and bonding of carbon black with the butyl rubber matrix, correlating the results with improved rubber tires (147). Hess has successfully used a combination of light microscopy, electron microscopy and microradiography to analyze the dispersion of pigments in rubber (195). He describes a simple, versatile nitrogen-freezing technique that permits sectioning of pigment loaded rubber stocks containing particles as small as 0.05 to 0.1 micron in size. Also described are simple tensile specimen holders that can be used to study the effects of strain on small rubber specimens in the x-ray and electron microscopes. I n a well referenced paper, LeBras (259) discusses in considerable detail the reinforcement of rubber by resins. Although making only occasional mention of the electron microscope, he indicates that this instrumental approach should not be overlooked, especially when attempts are being made to establish whether interaction of the resin with the rubber is chemical or particulate in character. Shvetsov et al. (416) have observed rubber sol, gel fractions and secondary filler structures in solutions of reinforced rubbers. They, too, conclude that good dispersibility, a low tendency to aggregate, and the ability of aluminate and silicate fillers to form network and chain structures are the most important factors in rubber reinforcement. Microstructures were related to the physical properties of polyvinyl chlo-
ride/acrylonitrile/butadiene copolymer systems (PVC/SBR), with and without talc fillers and mixed under different conditions (54). Optimum tensile behavior occurred when the degree of mixing established an interwoven fibrous texture. Undermixing or overmixing caused random dispersions of more or less large, discrete particles of rubber in a continuous polyvinyl chloride phase. Changes in fillers caused alterations in the fracture patterns. Also, the prior strain history greatly influenced the microstructure, particularly in those systems containing talc fillers. The effects of the heat treating of butyl vulcanizates on their structure and dynamic properties were discussed by Payne (352). His conclusions were based primarily on structural and particle dispersion information supplied by light and electron microscopy. To study dispersions of pigments in rubber, Moscou (220) cut sections of frozen rubber 1 to 5 microns thick. For examination with the electron microscope, the sections were thinned further by ion etching with activated oxygen while the specimens were supported on silicon monoxide sutstrates. This procedure caused less fragmentation of large agglomerates than did the normal ultra-thin sectioning method. ilndrews and Braden (12) used replicas to elucidate the physical mechanisms by which various additives afford ozone resistance to natural rubber. The elucidation of modes of crystallization of high polymers continues to occupy a prominent spot in electron diffraction and microscopy. Symons has examined (446, 447) the electron microscopical evidence for comparison to the fringed micelle concept of a tangled mass of chains, generally wandering in a random manner but occasionally including segments aligned to constitute small crystallites. Instead, he regards the more crystalline polymers, at least, as made up of discrete lamellar elements in which the molecular chains are accommodated in a rather regular folded configuration. On this basis the amorphous halo in diffraction patterns is ascribed to imperfections in the lattice rather than to the disorder of the fringed micelle. Polyvinyl alcohol single crystals were grown from solution by Tsuboi and Mochizuki (457). Holland and coworkers (203) have reported on the crystallization of polyacrylonitrile from dilute solutions of propylene carbonate in the form of ellipsoidal platelets, approximately 100 -4.thick. By electron diffraction, it has been shown that these units are single crystals and that the polymer axis is nearly perpendicular to the plane of the platelet. Manley (279) has grown single crystals of cellulose triacetate by supercooling dilute solutions in mixtures of nitromethane and n-butanol. The crysVOL. 36, NO. 5 , APRIL 1964
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tals are approximately square in shape and they thicken by spiral growths centered on screw dislocations. The molecular chains are perpendicular to the lamellae which are about 180 A. thick. Evidence is presented that would seem to indicate that the chains have assumed folded configurations in the lamellae, despite their stiffness. Crystals of cellulose triacetate were hydrolyzed and recrystallized to produce crystals of cellulose (280). However, in a nonaqueous medium amorphous cellulose was formed. His results suggest that hydrolysis is accompanied by a disruption of the cellulose triacetate lattice and that the subsequent recrystallization as cellulose is caused by the presence of water. Kargin et al. (230) have used electron microscopy and diffraction to investigate the morphology of single crystals of polystyrene and polypropylene. They find that the chains fold and form crystals of these polymers which are similar to other crystalline polymers that have been previously studied. Holland and Lindenmeyer (202) observed the morphology and made quantitative measurements of the crystallization of a series of sharp molecular-weight fractions of linear polyethylene. Their observations should be of value in verifying the theories of polymer crystalline growth mechanism. Electron microscopical studies were made by Ossini, Marchese, and Mazzarella (341) on polyethylene single crystals concerning: the influence of crystallization temperature on crystal habit and the effects of solvent etching on formed crystals. Hirai (199) presented data substantiating that the origination of screw dislocations on lamellar polyethylene single crystals, precipitated from solution, was caused by interference or collision of two grown crystals. Hirai, Kiso, and Yasai (200) utilized the rather unique method of tearing single crystals of polyethylene in order to study this texture. The influence of the structure of crystalline polymers on physical properties is shown by the work of Salovey (402). He found that the effects of irradiation by high energy electrons on crystalline polyethylene differed depending on whether crystalline lamellae were in contact with one another or separated. Keller and coworkers continue their excellent work on the growth and structure of crystallizable polymers (220, 235). A related paper by Bassett, Keller and Nitsuhashi (27) concerned the crystallization of low pressure polyethylene from xylene a t concentrations as great as 507& At high temperatures, axialites (so-called because they consist of layers or layer packets splaying about the axis) are formed. Viewed along the 188 R
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axis, the axialites appear to be sheaving in the form of hexagons or fibrous ovals perpendicular to the axis. I n some projections the axialites appear to be single crystals, in other projections incipient spherulites. Examination of the fine structure has shown that physical cohesion exists between the 100 A . terraces forming a layer packet on a 1000 A. scale. The layering is attributed to the interlamellar ties referred to above. The tying together of otherwise chain-folded lamellae is considered a plausible link between crystallization from dilute solution and from the molten state. In another investigation (235) the chemical reactivities of chlorine with solution and melt-crystallized linear polyethylene are explored along with molded polyethylene films. Differences in chemical reactivities are explained in terms of differences in structure. Taking a page out of the biologist's book, Spit (220) used phosphotungstic acid to delineate the fine structures of single crystals and spherulites. From the metallurgists, Bassett has adapted the technique of the detachment replica to strip a surface layer from polymer crystals (220) for study by electron microscopy and diffraction. The study of the spherulitic structure has progressed to include not only the special spherulites grown in the laboratory but also those that form in commercial polymers. Among natural polymers dndrews (220) has observed spherulitic structures in natural rubber films. He cast uncrosslinked films, 1000 A. in thickness, held them a t 26' C. for several hours and then examined them on the cold stage of the Siemens Elmiskop I. The spherulites consisted of central patches and filaments as indicated by electron microscopical and diffraction techniques. Employing replicas, Bailey (2f) has related the microstructural characteristics and physical properties of polyethylene and polypropylene. The study included polymer powders and molded pads. Differences in the spherulitic structure of polyethylene and polypropylene were observed, as well as the effect of molecular weight on spherulitic structures of both polymers. Using a fracture method, internal lamellae in bulk polyethylene have been revealed by Anderson (1, 9, 220). These lamellae attain a maximum thickness of about 250 to 300 A. An excellent, unified view of the present state of the knowledge of polymer crystallization, drawing heavily on work done in electron microscopy and diffraction, has been presented by Lindenmeyer (267). It is Lindenmeyer's view that crystalline polymers may be considered either as a two-phase or a single-phase system, depending on
how many independent variables are needed to characterize the properties of the system. Also, a hypothesis of continuously varying crystal habit is presented in which the prevailing crystal habit is attributed to the relative values of three nucleation rates. Continuing his earlier work (246) on the methods of studying heterophase conditions in resinous materials, Kronstein (247) presents further basic experimental approaches for inducing and studying desired phase formations and modifications in simple polymers, as well as complex polymer-metal systems. Other studies of complex systems are presented by Hock (1, 201 ), who showed electron microscopically that the rubbery and rosin-based components must interact to form two mutual phases in order to obtain optimum pressure-sensitive adhesion. Hock used 10% gelatin in water to make his primary replicas. So did Botty (1, 52) to study various heterophase systems, including a thermoplastic material of high-impact strength. By comparing results by replicating fracture surfaces of cold-embrittled specimens and of specimens at room temperature and by comparing the techniques of fracturing, microtoming and smearing into thin films, he established the validity of the technique of brittle fracture. Erath and Robinson ( I , 140) used both techniques of microtomy and brittle fracture to study several kinds of molded thermoset resins. They conclude that the micelle in the fundamental unit involved in the strength of thermoset resins. Wohnsiedler (1, @9), using well established replica techniques, concludes that both the macromolecules and their aggregates are meshed in a complex network. Spurr and Niegisch (430) have studied wet stress crazing in thermoplastic resins and have concluded that there is molecular cleavage. Coulehan, who is engaged in a series of studies (19, 96) of wet stress crazing, concludes that such crazing is a result of disenta'nglement of molecular domains. A symposium on the morphology of polymers a t Los Angeles (1) and another on resinographic methods a t Atlantic City (fg),both organized by Rochow reflect the widespread interest in the microscopical approach and the need for coordinating techniques with application. iilthough the many excellent papers also represented other instrumental approaches besides those offered by the electron microscope, the highly functional role of the latter was clearly indicated. The threshold of interest was sufficient to result in the formation of an ASTM Committee, E-23, on Resinography, whose prime function is to organize and promulgate information of mutual interest on all aspects of resinous, polymeric, and plastic materials.
FIBERS
This section on fibus is a review of some useful contributions that have been made by niicroscopists investigating natural and synthetic fibers having practical end uses. Fibers of this kind are commonplace for they can be found in fabric:, papers, cordage, sutures, reinforced plastics and elastomers, and a host of specific items. While their properties and uses are well known, many questions concerning the relationship of their complex structure to their unique or specific properties remain unanswered. Nat,ural fibers are known to be extrernely complicated entities (390). Synthetic fibers, once thought to be homogeneous, have been recognized as being coriposed of primary and secondary structures (31, 98, 229, 400). There are also wide varieties of chemically modified physically treated, delicately coated and intimately coordinated fibers (55, 66, 57, 118, 390, 391,433, 51 6). When .:hese complexities are added t,o those or integration and fabrication, i t become:i evident that an investigator must analyze his problem and plan his microscopical research with due regard t,o the number and relationship of molecular associations present, using a classification scheme such as that proposed by Rochow (387, 388, 389). I n t,he realm of natural fibers, Preston (220, 372) deftly uses the electron microscope as the tool for Tiisibly separating plant cells into their component parts in order to more completely understand their individual modes of growth and function in the plant cell natural environment. One of his prcmvocative findings is t,hat many plants ui ilize sugars other than glucose in the synthesis of the crystalline cell wall constituent. In general, the microfibrils are responsible for the strength and toughness of the cell wall, though the manner in which they are arrayed vayies widely from plant to plant. They may be highly oriented in lamellae or, in a few cases, randomly arranged. However, microfibrils are by no means found in all plant cells. Preston has examined some in which no true microfibrils were observed. Instead, the walls appeared to consist of granules or, perhaps, heavily incrusted short rodlets A paper of technical significance to the pulping industry is Liese's (266) explanation of the relatively unknown layer between the ligcified cell wall of tracheids, fibers and vessels of soft and hard woods and the cell lumen. This layer, known generally as the tertiary wall, is covered by a chemically resistant, so-called warty membrane that consists primarily of dehybrids of the protoplasm. Consequently, it is probable that the gels that cause trouble in pulping or viscose making operations may consist of insoluble warty mem-
brane fragments. Since the warty membrane and tertiary layer influence diffusion of pulping liquids from the cell lumen into the cell wall, it is conceivable that their structures contribute to the different behaviors of wood species in pulping. C8tB and Krahmer (95) have elucidated the permeability of coniferous pits by sectioning pieces of pine treated along the grain with India ink, applying a vacuum to one end and then determining the effects of various parts of the transport system of pine by the location of the India ink. Sachs, Clark and Pew (400, 401) stained lignin with p-(acetoxymercuri) aniline to show its location in wood cells. The greatest lignin density was found in the middle lamella, the primary layer and the Sa layer. Kardrop (475) discussed the anatomy of wood and the importance of the structural features of pits in determining the path of penetration of pulping media. The penetration was found to be related to specific sites of attack which varied according to the chemical or semi-chemical nature of the pulps. Morehead (311) describes a technique that permits the sectioning of single pulp fibers. He can select given fibers and even specific areas of fiber for cross-sectional examination. For example, in the study of the viscose reaction on pulp fibers, he demonstrated that the structure of undried wood pulp is distinctly more opened-up by the reaction than is dried wood pulp. The nonhomogeneous reactions encountered in the viscose process are illustrated by two pulp fibers that are side by side: one fiber is swollen almost t o the point of disintegration, the adjacent fiber has hardly been affected. Morehead has provided the industry with a valuable tool to study and understand the reasons for heterogeneous reactions. Papers produced from natural and synthetic fibers have also been examined by electron microscopy in conjunction with other techniques in order to determine the relation of the structural units to the properties of the whole sheet. Felton, Botty and Clark (144) have demonstrated the usefulness of replication techniques for the study of the fiber network and fiber surfaces in intact fiber sheets. Buchanan and Washburn (65) have used the scanning electron microscope for the purpose of relating fiber conformability and the extent of external fibrillar connections between fibers with the tensile properties of formed hand sheets. Using several methods Jayme (224) elucidated the structure of fibers with respect to the behavior characteristics imparted to paper and has propounded a thesis to explain the influence of fiber shape factors on the properties of paper. Cotton continues to receive a great deal of attention from the Southern
Regional Research Laboratories. deGruy et al. (115) investigated the effects of abrasion on cotton. They observed patterns of damage for a variety of experimental conditions and have offered a physical explanation for the differences in failure characteristics between uncrosslinked and crosslinked cotton. Rollins, Moore and Tripp (391) have examined crosslinked cotton, correlating changes in fine structure with differences in breaking strengths, elongations, and dry and wet wrinkle recovery. Styrene monomer that was reacted with cotton by exposure to a cobalt-60 source was examined electron microscopically by Demint et al. (118). The polystyrene was found within the growth rings of cotton. The radiation induced interaction improved the elongation a t the break, increased the resistance to wetting and reduced the stiffness of the cotton. The fibers became thermoplastic. Physicochemical studies of the cellulose-DMEU reaction, with particular emphasis on the catalyst, have been conducted by Ziifle, Berni and Benerito (515). Kaeppner (229) correlated the morphology of three disintepr- ., fibers with their degree of crystallinity obtained from x-ray diffraction data. He concluded that the observed fibrillar nature of the Fortisan was consistent with its high degree of crystallinity. Functional units derived from high wet modulus and tire fibers were observed to consist of flat ribbon-like particles near the fiber surface and short equant particles, respectively; both fibers had lower crystallinity. The significance of these findings was then considered in relation to surface structure, especially in the case of Fortisan where the longitudinal striations seem to be an expression of the underlying structure. Ultra-thin sections of heavy-duty cord rayon fibers, examined by Berestnev and Kargin ( S I ) , have indicated that self-reinforcement effects of macroformations of anisodiametric shape account for the fiber mechanical properties. Morehead (138) has designed a method for the selection and longitudinal sectioning of fiber crosssections for studying the structural elements along the fiber axis. Dlugosz (220) has done thorough experiments designed specifically for electron microscopical study to clarify the nature of the bonding mechanism of multicomponent adhesives to the fiber and to the carcass compounds used in the manufacture of tires. Scott ( 4 1 1 ) examined the crystalline structure of synthetic fibers by combining the techniques of electron diffraction and darkfield electron mirroscopy. Electron diffraction patterns from thin layers peeled from drawn fibers indicated the structural variations that occur within a fiber. These V O L . 36, NO. 5, APRIL 1964
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methods were then applied to drawn polyethylene fibers to reveal crystalline structures that had a n average size and distribution predicted from x-ray diffraction and light scattering studies. Along the same lines Harris (220) has investigated the crystallinity of nylon 6,6 filaments after annealing them in steam at 46 lb./sq. inch for 1 hour. Nylon filaments were first examined by selected area electron diffraction and certain reflections were then chosen for texture investigations by dark-field. Harris prepared his samples differently than Scott. Harris prepared thin sections using a novel arrangement of the filaments to reduce molecular reorientation to a minimum. The filament axes were normal to the top surface of the knife, hence approximately parallel to the stress set up by the knife. By darkfield electron microscopy no deductions could be made concerning the dimensions of the crystalline regions in the axial direction. However, at right angles to the fiber axis the crystallites were observed which measured approximately 0.1 M. Stamm et al. (433) combined electron microscopical studies of ultrathin fiber cross-sections with other methods to provide proof of the complete penetration of grafted chloromethylstyrene through polypropylene fibers. Structural variations of the polypropylene fibers induced by the grafting process were also reported. Craig, Knudsen and Holland (98) integrated several techniques to study the fine structure of acrylic fibers and the effects of varied spinning conditions on their morphology. Gross differences among the samples were observed light microscopically depending on whether they were wet or dry spun, and the conditions under which they were extruded, drawn and collapsed. Variations encountered were different degrees of collapse, cross-sectional shape, and the number and shape of the large voids. Despite such gross differences, collapsed wet and dry spun acrylic fibers, regardless of spinning conditions, were consistently found by electron microscopical studies to have a fine fibrillar, and void structure. The fibrils and voids were each approximately 200 A. in diameter. For wet spun samples, this structure originated during coagulation and was traced through stretching and collapsing. On the other hand, the fine void-fibril structure in dry spun samples only became evident after the orientation step. Bradbury, Rogers, and Filshie (55,66, 57) have presented a series of articles on the fine structure of wool and its chemical modifications. Their contributions to the knowledge of wool are enormous. Here is an exceptionally fine study in which many techniques avail190 R
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able to the microscopist are sensibly put to use to exploit the potential of microscopy in fiber research. It is a series well worth studying as a guide to a planned integrated microscopical examination of any fiber. Sanders (403) presents a n unique method for the direct replication of fiber surfaces. Newman (329) has shown that the point projection x-ray microscope, a n instrument closely allied to the electron microscope, can be of considerable value in examining fabricated polymer articles, either filled or unfilled, such as fabrics, laminates, foams, etc. Its principal values lie in its considerable depth of field and the fact that structure can be examined in situ in many instances. Although not dealing specifically with instrumental techniques, a series of articles published by Hearle should prove to be a valuable aid in the interpretation of results in terms of structure and properties of fibers (184, 185, 286). I n Part I (184) he discusses the concept of the fringed fibril structure and proposes a modification to account for long periodicities. He concludes with a survey of the structures of different types of fibers and polymers. In Part I1 (185) the crystalline growth within a fiber under different extrusion conditions and the effect of drawing and orientation on the crystalline structure are considered. Part I11 (186) deals with the mechanical properties of some fiber structures, in particular, the extension of plant fibers in terms of a spiral arrangement of crystalline fibrils embedded in a noncrystalline matrix. Hoseman’s (208) article on the type of crystallinity present in fibers is similarly good reading for the electron microscopist. By employing the fundamentals of structural analysis, he had defined the structure of fibers. Hoseman describes a paracrystalline lattice, draws conclusions and postulates theories which are relevant to the interpretation of electron diffraction patterns and darkfield electron microscopical images of fibrous materials, both natural and synthetic. A perusal of the individual works contained in the above section shows that electron microscopy continues in a position of eminence as a means for research into the structure of fibers and fibrous materials. The challenge to fiber microscopists is to develop methods by which microscopes may be used to their fullest potential for fiber research, and to apply interpretative skills to the data collected in order to determine how the elements of fibers are arranged and how these structural arrangements affect inherent properties. METALS
Metals are under intensive scrutiny by electron microscopists. The orderly
development of the study of the structure and properties of metals has been stimulated by new preparative techniques for direct observations of imperfections in ‘crystals. The text, “Transmission Electron Microscopy of Metals,” by Thomas (461) reviewed Lewis (266), is an excellent reference source as well as a medium of instruction concerning principles, practice and application of electron microscopy and electron diffraction technology. The chapter on the preparation of specimens is well referenced and discusses a t least ten techniques for obtaining suitable, thin crystalline specimens. Electropolishing of the bulk specimen, cleavage, microtomy, and vacuum deposition, are just a few of the techniques that are described. Another book, the “Direct Observation of Imperfections in Crystals” (125),edited by Newkirk and Wernick, contains valuable information derived from the application of diverse instrumentation, including field electron emission, field ion emission, and transmission electron microscopes. The published abstracts of I F E M S and EMS.1 (158, 2220) and the numerous technical bulletins, usually available on request from industrial firms, add to the availability of technical information. For example, the Norelco Reporter published details about a n electron diffraction technique for identifying oxide layers on crystal surfaces (458) and a description of the aspects of x-ray optics as they apply to electron probe microanalysis. Design features of a hot and a cold stage (73, 92) and a replica technique for studying metals appeared in the RCA Instrument News. An electron microscopical study of precious metals has been published in a technical bulletin (412). The .iSTM Special Technical Publication No. 317 (18) recapitulates progress in metallurgical techniques and instrumentation and contains a first progress report by an ASTSI group on electron transmission microscopy of metals. Carroll (74) has given good examples of the application of the electron microprobe to metallurgical problems such as phase identification, analysis for inclusions, and evaluation of corrosion, oxidation, and diffusion in the solid state. Watts (486) has described how metallic surfaces can be ion etched, chemically reacted, and finally imaged by employing the secondary emission microscope. The concept of the dislocation defect in metal crystals and its theoretical and practical importance were presented by Mott (515) in his published lecture on atomic physics and the strength of metals. Of the many imperfections that occur in metals, the dislocation defect is of prime importance because of its effect on the practical utilization of metals ($54, 509). Silcox (417) has defined the principal imperfections in
crjstals, such as the dislocation, the grain boundary, and the stacking faults, and then describes how transmission electron microscopy can be used to identify and study these defects. Image formation of lattice iinperfections, contrast and electron absorption effects have been considered from a theoretical point of view (179, 280, 281). Polyphase systems as well as pure metals have been studied. Marcinkowski and Miller (282) reported on defect substructures, consis,ting mostly of faults in the (100) and the (010) planes of crystals of the FE-Cr sigma phase, which they believed were developed when the bulk metal transformed from the ferrite to the sigma phase. Their 34-page study was supported by numerous electron micrographs and schematic drawings. Turkalo (220) studied deformation twins in siliconiron. .In electric sprirk technique was used by Gemperle, Rozsival, and Sestak (157) to cut F'e - 3.2% Si alloy single crystals that hrtd been deformed by bending. Surfaces cut parallel to the slip plane were further thinned by polishing and by graddal anodic etching for their study of the distribution of dislocations in the d i p bands. The various types of loops,, observed microscopically, were schematically drawn. Dislocations resulting from the allotropic transformation of pure iron in bulk and in thin foils were investigated by Yamashita and Trmeda (508). Thomas and Hren (220) observed heterogenous precipitation in a n Al20y0 Bg alloy. Nucleation, growth and dissolution of the hexagonal y1 phase was studied by varying the thermal conditions of the t,pecimen in the electron microscope with a hot stage. Sequential changes were recorded by cine photography. Watanabe, Hayashi, and Hayashi (478) examined a similar alloy system in order to study changes that were associated a ith age hardening properties. The significance of the observed dislocation loops, stacking faults, and precipitates were discussed. Attempts are made to thin the specimen so that it retains the properties of the bulk specimen. This is not always possible to achieve. Wilsdorf and Kuhlmann-Wilsdorf (1'20) reported that the shape of dislocation lines in aluminum crystals varied, depending on whether deformation was induced in the thinned film or in the bulk material. Further variations were observed when the bulk specimen was deformed a t 4" K. compared with one deformed a t room temperature. The presence of smooth, tangled, and prismatic dislocations in the respective samples was explained on the basi,; of different dislocation-forming mechanisms. In a related study the unexpected cross-slip observed in quenched gold foils was explained by Kuhlmann-Wilsdorf,
Maddin and Westdorp (248) on the basis that there were fewer vacancies in the thin foil compared with the greater number present in the bulk material. Thus, slip occurred in the foil, whereas dislocation entanglements occurred in the bulk specimen. The significance of the three types of dislocations observed in single crystals of BCC molybdenum, deformed over the range 4.2" to 300" K., was discussed by Lawley and Gaigher (258). Wilsdorf and Schmitz (494) made a n extensive study of dislocation tangles in the easy glide range of aluminum. Zirconium is a n important nuclear material. The dislocation behavior of vacuum annealed, electrolytically thinned, zirconium foil was investigated by Howe, Whitton, and McGurn (212) who thermally induced mobility and interaction of dislocation lines by varying the intensity of the electron beam. Dislocation traces, which persisted for relatively long durations compared to the short-lived traces in aluminum and stainless steel, suggested the presence of a tenacious film on the surface of their test specimen. Bailey (220) showed that impurities such as oxygen and nitrogen affect the arrangement of dislocations in deformed polycrystalline zirconium, producing hardness. Increased hardness of his test specimen was believed t o be associated with restricted dislocation movement. Ruff (395) developed a procedure for etching thin, singlecrystal foils of copper so that etch pits and dislocations were simultaneously revealed. A relation between etch pits and dislocations was established although a one-to-one agreement did not exist. The possibility of other types of defects that might nucleate etch pits was also discussed. Booker and Stickler (220) observed domain formations in germanium specimens that were mechanically deformed and subsequently annealed. From the characteristic configurations formed in the test specimens it was suggested that the stacking fault energy of germanium is in the medium to high range. Regarding epitaxial growth and film formation, it was shonn by Sloope and Tiller (419) that the size of germanium crystals in films depended on the rate a t which the germanium was vacuum-deposited and on the temperature of the crystal substrate. Polycrystalline films formed between 300' to 350' C. ; single crystals between 450' to 700' C. Raltz (22) made a similar study, producing both polycrystalline films and single crystals of a Ni-17y0 Fe alloy. Vook (2200) prepared films of oriented single crystals of tin on cleaved NaCl substrates and examined them for dislocations. Morita (313) measured the Curie temperature and the specific electrical resistance of vacuum-deposited nickel film- as a
function of film thickness. He found that the Curie temperature of the thin films was nearly the same as that of the bulk material when the ratio of pressure to deposition rate is below a certain value. Materials exposed to high energy radiation undergo varying degrees of damage depending on the type of radiation and the organization and chemical composition of exposed material. The kinds of damage and the resulting defects that might become visible in the electron microscope were considered by Goland (220). From theory, it was considered that radiation damage could generate a variety of microscopically recognizable formations. Blank and Amelinckx (46) reported preliminary findings of defects in irradiated single crystals of UO,. Single crystals grown by condensation from vapor phase were cut and ground into discs and chemically thinned. Dislocation lines were observed and the interaction between dislocations and fission tracks were discussed. Fission fragment damage in UOs single crystals was also investigated by Koggle (138) who combined replica studies with direct observations of fission tracks for determining the location of the induced defects. Whapham and Makin (220,490)discussed two different types of tracks which they observed in single crystals of UO2 that were made by oxidizing and annealing uranium foils in the electron microscope. Studying fission fragment damage in molybdenate crystals, Izui and Fujita (222) also observed an interaction between dislocation lines and fission tracks. From this they deduced, as have others, that strong stress field around the tracks cause the slip motion of dislocations by elastic interaction. Their study was enhanced by tilting experiments and the evaluation of moire and electron diffraction patterns. Ruedl, Delavignette and Amelinckx (220) studied P t foils that were esposed t o neutron, y particle and fission fragment irradiation. Significant variations in the number of loop formations were believed to be dependent on the presence of nuclei produced by neutron irradiation in the variously treated foils. Piercy and Whitton (362) gave semiquantitative values for dislocation loop densities observed in pure aluminum and in Al-l% hlg alloy after fast neutron irradiation, and discussed the effect and significance of radiation-induced vacancies on the formation of dislocations in quenched or deformed specimens. Defect structures in neutronbombarded aluminum were observed by Bierlein and Mastel (37). Kelsch, Kammerer, and Goland (236) reported on fission fragment damage in an electrolytically thinned aluminumuranium fuel cell element. In this study, track-like markings, believed to VOL. 36, NO. 5 , APRIL 1964
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have been formed by a thermal spike mechanism, were observed only in the thinned, irradiated specimen, not in the bulk specimen which was irradiated and then thinned. Fission fragment damage in metals has been extensively investigated by Kelsch, Kammerer. and Goland (237), by Noggle and Stiegler (332), and others (220). Xeutron damage in molybdenum was studied by Bierlein et al. ( I % ) , low temperature oxygen ion damage induced in copper and Cu-Be alloy foils by Piercy and Gilbert ( I % ) , and electron irradiation damage produced in NaCl crystals during observation in the electron microscope by Hosomi and Meska (238). KC1 single crystals were exposed t o high and low intensity levels of electron irradiation in the microscope. From this extensive study, which included replication, electron diffraction dark field and hot stage techniques, Hibi and Yada (196) deduced a probable mechanism for coagulation of vacancies in electron-irradiated KC1 crystals. A vacuum-deposited germanium film which was heat-treated t o induce grain groMth was exposed t o two levels of neutron bombardment by Parsons, Balluffi, and Koehler (350). Characteristic defects were observed electron microscopically that were in general agreement with the size and number predicted by earlier investigators who used indirect methods of experimentation in the study of electrical conductivity and Hall mobility in neutron bombarded germanium crystals. An unusual effect was observed by Slooten and Sowden (420) when they irradiated aqueous suspensions of fissile oxides such as plutonium dioside and (Th, Pu)02and (Th, U)Oz. Under suitable conditions, fragmentation of the oxide particles occurred and a lyophobic colloidal system developed, presumablv as the result of surface action. The structure and magnetic behavior of metals are being correlated 11ith specific domain patterns by means of electron microscopy. Gondo (f 61) made a detailed study of single crystals of iron which were epitayially formed on the heated, cleaved (001) surface of MgO. Epitaxial growth was confirmed by electron diffraction esamination. He modified the Bitter technique to obtain a colloidal dispersion of very fine ferromagnetic particles. When the colloid was applied to the iron crystals, the outline of the domain walls became light-microscopically visible. A carbon extraction replica technique was used t o study the fine line detail of the Bitter pattern and its relationship t o the magnetic property of the sample. In a short note, Furuoya and Sasaki (154) discussed the dependence of size and temperature on the superparamagnetic properties of fine nickel 192
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powers which were made by vaporizing nickel in a low pressure argon atmosphere. The size of these particles increased with increasing argon pressure. At a pressure of 0.1 mm. of Hg the nickel particles were about 100 A. in size whereas a t 10 mm. of Hg the particles were about 300 A. in size. Knowledge of the direction of magnetism is necessary for the accurate interpretation of the domain configuration as observed electron microscopically. Wilson (495) has suggested that the light and dark areas associated with holes in ferromagnetic films, viewed by the off-focus technique, may establish the magnetic direction. Wade (472) offers further comments and suggestions based on his observations of contrast effects a t edges and cracks in ferromagnetic films. Spain and Puchalska (220) discussed the significance of line shifts and changes in line intensities in superimposed ferromagnetic Si-Fe films that were separated by a thin gold film. When one must know the intensity of the magnetic field on the ferromagnetic sample in the microscope, the procedure of Luborsky and Koch (269) may be helpful. The microscopist is relying more on thinning methods than on replica methods for studying metals, although in fractography (23, 440) replication is still the best method of approach for studying morphology unless one has access to a secondary emission microscope such as the Metioscope (38, 486). For the removal of a disperse phase, such as precipitates, for subsequent diffraction studies, the extraction replica method has advantages. However, in many cases, the surface replication techniques have been reduced somewhat to a secondary role. Pellier (220) has shown that a hardening intragranular precipitate in aged Inconel X was made visible by thinning the bulk nickel as well as by replication methods. Perhaps final selection of methods nil1 depend on the availability of proper electrolytic thinning reagents, skill, and time. Ebben and Lawson (135) applied a chromium-shadow, carbon replica technique for a better understanding of the changes in tin plate alloy structure with respect to the electrochemical and corrosion resistance properties. Using an aluminum oside replica technique, Coates and Bohl (88) report on a study of an & 0 3 disperse phase on a hardened aluminum alloy. At first hand, one might not expect to resolve discrete A1203 particles in a replica material of similar composition. Hoaever, the dispersed .k1203 oxide particles became visible because the particulate oxide and the replica oside have different electron diffraction characteristics due to their different crystalline form, thus producing image contrast. Doherty and Davis (226)
developed and applied a replica technique for removing the h12O3 layer that formed over small pits on the surface of thermally treated crystals. From their excellent electron micrographs, crystallographic orientation associated with oxide formations over the pits, a t the socalled oxide-oxygen interface, was studied. The textures of anodized aluminum surfaces were investigated. A carbon replica technique was used to investigate dendritic inclusions in copper electrodes in an attempt to explain the breakdown of electrically stressed metallic surfaces in a vacuum (182). Carbonyl ion powders were found to have a multilayered, onion-like secondary structure which is believed responsible for its pronounced electromagnetic properties (357). Esquivel el al. (141) used carbon replicas to evaluate surface textures of tantalum and platinum substrates and the texture of lithium which was deposited on these substrates. In this study, which concerned selection of suitable lithium films for the p-n reaction in the Van de Graaf generator, it was found that tantalum and platinum served as better substrates than carbon. The decoration technique (97), which may involve the electroplating of the cleaved surface of a crystal to make crystal imperfections visible, also relies on a replication method (295). Metal whiskers are well suited for examination by field emission techniques especially when the difficulties in mounting have been overcome (297). When the whiskers are small enough they can be viewed by brightfield and darkfield and by diffraction contrast techniques to study extinction line contours (25‘7). These are but a few of the many investigations in the metallurgical field which exemplify timely applications of electron microscopes to complex problems. CRYSTALS AND CERAMICS
In addition to metals and their oxidized surface layers, the industrial electron microscopist is concerned with studies of an ever-increasing number of other crystalline materials. These include such diverse materials as sapphire and organized luminescent particles. Sapphire was examined by Voruz, Jewett, and Accountius (470) for dislocation defects as a prelude to investigations on the factors influencing the mechanical behavior in ceramics. Luminescent, rhomboidal particles, derived from a photosynthetic and luminescent marine flagellate, were studied by De Sa, Hastings, and Vatter ( 2 1 9 ) . The second “Chemistry Edition,” (502), compiled from international sources by Suito, exemplifies the diversity of organic and inorganic crystals that have been examined electron-optically. This
worthy complication of electron micrographs, which is part of the series, “The World through the Electron Microscope,” (502),published by Japan Electron Optics Laboratory Co., Ltd., is a valuable referencc source. Because of their industrial importance, the allotropic forms of carbon continue to be investigated. Evans and Phaal (220) found a greater number of dislocations in the Type I1 diamond than in the Type I, suggesting that the Type I1 diamond formed ai a lower temperature or cooled at a :lower rate in the presence of certain inipurities Well-annealed, cle,i\ ed platelets of pyrolytic graphite a ere examined by Tarpinian (449) who discussed the equilibrium grain texture and the socalled secondary recrystallization structures as uell as the nodular arrays that were produced by ion etching. Hennig (191) discusses vacancies and dislocation loops ob3erved in rinnealed graphite platelets, and the problems of relating these defects with cmsistent physical values for the energ.$ of vacancy formation. Several investigations of the effects of radiation on graphite were reported a t the Fifth International Congress (220). Elcbctron microscopy has been used to ascertain the changes in the size of soot particles, in a study, on the combustion of soot in a laminar soot flame, by Lee, Thring, and Beer (260). Walker (473) has reviewed some of the important forms of carbon; he emphasized their preparation and properties, and referred t o pert nent examples of instrumental application for evaluating structure. Gallium selenide is one of the materials which, in certain forms, can be easily cleaved into thin platelets and examined by tranc,mission electron microscopy. Basinski, Dove, and &looser (26) examined thin crystals, 500 t o 1000 A. thick, gro7n.n from the vapor phase, as well as clrhaved crystals, to study interacting partial dislocations and the associated n3twork of dislocation grids that formed. They discussed the contrast effect produced by dislocation dipoles contrtined in the grids. From studies of didocation ribbons, Price and Sadeau (3;Y4)concluded that t u o sets of stacking ’aults, corresponding to different energy levels, were produced in plastically deformed single crystals of nickel bromide. Blank and Amelinck.; (45) made direct observations of ferroelectric domains in thin crystals of barium titanate and Ilelavignette and hmelincks (117 ) observed antiferromsttic domain walls in cleaved, thinned single crystals of nickel oaide. The domain walls were depicted as interference fringes, similar to those observed in stacking faults on twin boundaries. which become visible due to differences in the diffraction conditions.
Stadler (432) etched BaTi03 crystals with HF and observed that small, flat mounds formed where a well-defined domain wall had previously existed An explanation for the probable formation of these mounds was given. Prismatic dislocation loops induced in golddiffused silicon were investigated by Phillips and Dash (358), who concluded that the loops surrounded a stacking fault in the silicon lattice rather than particles of gold coherent with the silicon lattice. An infrared micrograph of prismatic loops supplemented the transmission electron micrographs. I n their study of neutron irradiation of quartz, Teissmann and Sakajima (488) established a relationship between the total volume fraction of defect clusters and the fractional decrease in density induced by the neutron irradiation. h surface replica technique was used by Rau (377) to study the porosity, etching characteristics and general microstructure of extruded and sintered Be0. His examination of this material indicated that pore structure and etching characteristics were related to the hexagonal crystal structure of BeO. Electron microscopy was used by Spriggs, Brissette, and Vasilos (429)to characterize the microtextures of pressure-sintered magnesia, and by Vasilos and Spriggs (465) in a related study of the effects of pressure and temperature on the microtexture and density of pressed MgO and Xl2o3discs. Microscopical observations on the thermal behavior and decomposition of thin crystals of lead hydroxide, prepared by 51OW precipitation in alkaline solution, were made by Sole and Yoffe (426). I t was suggested that the formation of crystallographic cavities and nuclei may be a characteristic of thermally decomposed layered structures in which there is a fair degree of covalent bonding. Williams, Sinclair, and Koonce (493) found that electron microwere well suited for stry and morphology of thin mullite films, especially since individual crystals, which formed in the films under certain conditions of firing, nucleation and thickness, could be selected and analyzed by electron diffraction. The results of an electron microscopical and x-ray diffraction study of the early stages of paste hydration of certain cement compounds, with and without gypsum andlor XaOH, were reported by Chatterji and Jeffery (79). Paste components, identified by microscopical techniques a t different intervals of hydration, were listed in tabular form. Sterman and Xlarsden (437) studied the filler-silane interaction. Argyle and Hummel ( 1 5 ) reported that with the aid of the electron microscope, it has been shown that a stable liquid immiscibility exists in the system BaO-Si02 above about 1655’ C.,
a fact to be remembered when considering the equilibrium diagram for this system. Krinsley and Takahashi (246) applied electron microscopy to a geological problem, comparing the surface t,extures developed on quartz grains under known weathering conditions with textures produced artificially by sand blasting, ball-milling and pressuregrinding experiments. Criteria have thus been established for identifying sand grains from specific geological environments. The surface of consolidated mineral materials can be studied by electronoptical, reflection methods. X scanning microscope, which had a depth of field of about 50 microns, was used by Thornley and Cartz (453) to distinguish different’ phases, components and other details on the surface of ceramic moldments. Limitations, advantages and potential of the method were discussed. The electron microscope has been used in conjunction with nunierous studies of the structure of glass. Moran (309) used an extraction replica technique to show a silica-rich second phase in quenched CaO.SiOz glass and the associated crystalline and vitreous particles. Chen (82) made kinetic studies of crystallization in synthetic mica glass and relied on the electron microscope for certain aspects of the investigation. Stookey (443) reviewed the chemical and physical nature of common glass, and then elaborated on the unique properties of modern glass. He effectirely illustrated the conversion of amorphous glass to crystalline glassceramic in a series of electron micrographs. Charles (78) supported t’he study of the electrical properties of lithium silicate glass with electron micrographs that showed significant, structural changes induced by partial dwitrification of phase changes. Much research has been done on synthetic organic fibers, but relatively few electron microscopical studies have been made of synthetic and natural inorganic fibers. Huggins (216) has compiled and described electron micrographs representing twenty inorganic fibers, many of which were prepared synthetically and have refractory characteristics of possible industrial concern. Zarzycki and Mezard (510)have made direct observations of the structure of thin glass filaments which they spun by an ingenious device, inside the microscope. Thus it was possible for them to examine the structure of glass fibers before and after contamination in the atmosphere. The textures and paracrystalline domains which they observed were discussed. -4study such as this appropriately reflects the brilliant efforts being made today to derive useful information through diverse applications of the electron microscope. VOL. 36,
NO. 5 , APRIL 1964
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ELECTRON DIFFRACTION
Electron diffraction techniques have been applied to investigate the molecular structure of organic molecules in the vapor state (142, 231, 393) and in thin polymeric films (233, 240). Low energy electron diffractim methods (6 to 600 volts) (277) have been used to study surface phenomena, and the advantages of high energy techniques (1200 kv.) for the examination of relatively thick foils have been reported (134, 220). The principles and advantages of low energy electron diffraction were described by MacRae (Wry), Hagstrum ( l 7 0 ) , and Lynch (274) who emphasize the sensitivity of the method for characterizing significant changes in structure at the surface of materials. Lander and Morrison (255) have studied the oxidation and reduction of silicon by low voltage electron diffraction and have also considered scattering factors and other aspects of the method (256). The above are special applications of electron diffraction. Most electron microscopists apply selected area diffraction or, with the aid of an adapter, (38) reflection electron diffraction techniques at voltages between 50 and 100 kv. in conventional electron microscopes. The combination of electron diffraction and microscopy is particularly useful for studying epitaxially deposited thin metal films on crystal substrates heated over a wide temperature range. Pundsack (376) in his study of electrical properties of thin Ge films clearly illustrates by means of electron diffraction patterns and corresponding micrographs the transitions of vacuumdeposited germanium films from the amorphous to the polycrystalline and single crystal state as substrate temperatures varied. There is no doubt that best single crystal orientations occurred between 550' and 570' C. The combined procedures of electron diffraction and microscopy have been used by others to investigate epitaxial growth (22, 419), the dendritic crystallization of an amorphous Ge-Te alloy (492), the orientation of an Altos surface layer which bridged etch pits in an aluminum foil (126), and the crystalline nature of lithium films ( 1 4 1 ) . Schoening and Baltz (408) studied twinning in annealed single crystal NiFe foils by these methods. The sharp Kjkuchi patterns, observed by Vook (469) indicated a very high degree of crystal perfection in ultrahigh vacuumdeposited tin films. Yamaguchi (506) has established a mathematical relationship between the mean weight of colloidal particlqs, such as MgO, ZnO, and Tho?, and the diffuse nature of t>heir electron diffraction patterns. He observed that when the particles were examined with an 194 R
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electron beam having an associated short wavelength (approximately 0.028 A.) diffuse ring patterns or reflections were produced. This was believed to be caused by recoil of the particles from collision with fast moving electrons, giving rise to mechanical fluctuations analogous to a Brownian motion. Thomas (452) gives a procedure for correlating slip traces visible in a micrograph of an aluminum foil with a selected area diffraction pattern of the specimen so that the corresponding slip planes in the foil and the foil thickness can be determined. The theoretical basis of electron diffraction structural analysis has been discussed by Cowley (280) and general treatment given by others. Corrections that can be applied to electron diffraction data have been Ieported by Bonham and Ukaji (50), who provide a mathematical treatment for dealing with the aspects of the atomic electron scattering factor, and by Bendler (620) who encountered anomalous lattice spacings in single crystal electron diffraction patterns of si!icon. The theory regarding the multiple reflection phenomenon was diwussed by Heattie (29). Kuwabara (250) used an electron filter technique for measuring the intensities and profiles of electron diffraction patterns and was then able to obtain information concerning the elastic and inelastic scattering effects associated with the specimen. Grigson et al. (260) have added an electron velocity analyzer to a scanning electron diffraction apparatus to permit the direct display of electron intensity profiles, along the radii of diffraction patterns, corresponding to particular energy groups. According to Davey and Deiter (109) glass micioscope slides as received from the manufacturer may contain traces of contaminants that could produce ambiguous electron diffraction patterns and confusing results when the slides weie used as substrates for studying deposited thin crystalline films. Structures in residual contaminants on glass, the possible source of contamination and a corrective cleaning procedure were discussed. Domain and general structural studies have been made on many materials. de Keijzer and Burgers (116) decomposed a ferromagnetic, supersaturated Au-Ni alloy. A Lorentz shift of the diffraction lines was observed which reached a maximum for a certain decomposition state. They suggested that the shift maximum is related to a maximum in the coercive force of the decomposing alloy. An analysis of an antiferromagnetic substance, a specially prepared NiO-Ni hlend, was examined by powder transmission electron diffraction techniques, by Yamaguchi (504). When the diffraction pattern of this powder was compared with the
normal NiO pattern, line intensity anomalies were observed which were correlated with the antiferromagnetic properties. In another experiment Yamaguchi (506) passes an electron beam through very small holes in a crystal of magnetite which is positioned between two razor edges serving as north and south poles. The shift in the electron diffraction patterns that is produced by the Lorentz force of the magnetic induction in the FeaOl crystal made it possible to determine the magnetic(l11) axis of the crystal. Using the technique of epitaxial growth, the superlattice associated with annealed Ni siugle crystals was studied by Alessandrini and Freedman ( 2 ) . The diffraction patterns indicated the presence of both Ni and N O . Forbidden reflections and split Xi spots were observed a t annealing temperatures in excess of 400" C. The significance of the disappearance of all extra reflections when the sample was heated above 650" C. was discussed. High temperature studies of anti-phase domains in Cu-Au compounds were made by Sato, Watanabe, and Ogawa (404), Toth and Sat0 (Q56), and Yamaguchi, Watanabe, and Ogawa (507). Reflection electron diffraction techniques were coupled with replica studies of the microstructure of electropolished iron single crystals during vacuum-annealing and hydrogen treatment a t 800" C., in an extensive investigation by Sewell, Brewer, and Cohen (413). Diffraction patterns produced by the crystal surface indicated the existence of relatively large, flat surfaces which were well suited for the examination of thin overgrowth-films or other studies requiring flat iron surfaces parallel to low-index crystal planes. Waber, Olsen, and W7hyte (471) describe their experiments for studying the removal and formation in vacua of oxide films on uranium and plutonium. From the electron diffraction data, they concluded that these metals oxidize even when polished in vacua of the order of IO-' torr. Dobson and Wilman (125) have studied the changes that occurred in polycrystalline salts of the CsCl structure type on exposure to compressive forces of 75 tons/sq. inch. Reflection electron diffraction patterns showed that crystallographic slip in the (011) plane produced a final orientation that was in agreement with the observed compression texture. The several crystal habits of the long chain alkane, n-CggHm, crystallized from solution and from the melt, were reported by Khoury (240) in his combined electron diffraction and microscopical study of this material. Many organic polymers in the form of single lamellar crystals have been examined (166). To the list of characteristic electron diffraction patterns already ob-
tained that of cry3talline polyvinyl alcohol can now be added (457). There is every reason to believe that the list will continue to groh. CONCLUSION
The members of EMSA, approximately 1280 in number, were informed that the Society would like to compile a listing of all courses given in the field of electron microscopy. This will require a cooperative effort by EMSA members, from which many will receive benefits in return. Courses in electron microscopy are knomn to be scheduled in the East a t Drexcl Institute and a t Cornel1 University, snd on the West Coast a t the University of California at Berkeley, but there are probably others of which we are not aware. A complete listing would indeed Ee a convenience. In addition to the foregoing texts and special publications cn electron microscopy mentioned in this review, several other treatises have appeared which may be of value. The text “Introduction to Microscopy” (497), by Wischnitzer, provides a comprehensive treatment of electron optics (304). This text, coupled with Kay’s book on “Techniques for Electron Microscopy” (450) will be of particular value to those who have only a cursory knowledge of electron microscopy and wish to broaden their background on this subject. For the polymer scientist:;, there is the text, “Polymer Single Cry3tals” (156) by H. Geil, which is based in a large part on electron diffraction and microscopy. Geil presents the awailable theoretical and experimental information on the morphology of polymers and the effect of the morphology 011 properties. The relatively expensi ve, two-volume “Trait6 de Microsccpie Electronique” has been considered by Cosslett (03) to be a n outstanding accomplishment. Volume 1 of a new publication, “Journal de Microscopie” (826), appeared for the first time in April, 19152. A timely book by L. S. Birks, “Electron Probe Microanalysis, ” (48) consolidates a description of microprobe instrumentation, the methods of analy3is and the various factors that have to be considered for obtaining useful data. The [‘Proceedings of the Syniposium on Cytochemical Progress in Electron Microscopy” (375) Oxford, England, July, 1962, were published by the Royal Microscopical Society as part of the Society’s Journal. The advances as well as the problems in this area of difficult microscopical endeavor can be appreciated from this collection of excellent papers. The Society convened for its celebration of “The Tercentenary of the Microscope in Living Biology” at the National Institutes of Health, Bethesda, Md., thereby establishing a precedent for holding meetings of the
Society outside the United Kingdom. It is understood that the scientific proceedings of this auspicious and productive occasion will be published in the Society’s Journal. We are reminded that Section 6 of the “Bulletin Signalktique” (67) for 1964 contains a “Bibliography of E l k t r o n Microscopy” that has been developed for EMSA. Perhaps this bibliography will substitute to some extent for the excellent “International Bibliography of Electron Microscopy” which was compiled by the New York Society of Electron Microscopy (NYSEM) until 1961, but was discontinued thereafter. Volume I, covering the period 1950 to 1956 with 4,054 references, and Volume 11, which lists approximately 12,000 references for the years 1956 to 1961, are available from NYSEM; prices have been quoted (333). The International Federation of Electron Microscopical Societies has sponsored three Regional Conferences and two World Congresses. The next Regional Conference will be held in Prague, Czechoslovakia, from Aug. 26 to Sept. 3, 1964. The Fifth International Congress for Electron Microscopy, which in effect was the second World Congress, was held a t Philadelphia in 1962 with EMSA as host, and the vital statistics of this meeting have been cited (’7). As of Aug. 1963, EMSA still had copies of the two-volume proceedings of this meeting which were available to EMSA members at a special price. The 44 sections contained in these two volumes manifest what may be termed the horizontal and vertical development of electron microscopy, for there has been tremendous growth and diversification in both instrumentation and application-a condition that can create additional problems for microscopists who are sometimes reviewer s. ACKNOWLEDGMENT
The authors gratefully acknowledge the assistance of their colleagues at the American Cyanamid Co. American Viscose Div. of F M C Corp. who helped with the many details involved in preparing the manuscript. I n particular they thank I. C. Piscopo and Esther Chasan, both of American Cyanamid Co., for the literature search and for critical reading pertaining to certain sections of the manuscript. The authors also thank the members of the administrative staff of both corporations for permission and encouragement to write this review. LITERATURE CITED
(1) ACS Symposium on the Morphology of Polymers, Los Angeles, Calif., April 4-5, 1963; Preprinted by Div. of Or-
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