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Baudler, M., Z . a m r g . u. allgem. Chem. 279, 115 (1955). Baudler, M., Z . EEektrochem. 59, 173 (1955). Bernstein, H. J., Allen, G., J . Opt. Soc. Anier. 45, 237 (19551. Brandmuller, J., Optik 12, 389 (1955). Brandmuller, J., Moser, H., Ann. Physik 13, 253 (1953). Chem. Eng. .Vews 33, 4232 (1955).
Chiuroglu, G., Doehaerd. T., Naquestiau, h.,BUZZ.soe. ehim. Belges 63, 470 (1954).
Geiseler, G., Kaufhold, R., Runge, F., Erdol
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Goubeau, J., Jahn, E. L., Kreutrberger, d.,Grundniann C., J . P h y s . Chem. 58, 1078 (1954).
Heilmann, R., Arnaud, P., B u l l . soc. chim. France 1954, 1257. Jorge, M.del P., Matutano, J. R. B., Anales real soc. espafi.f i s . u qubm. ( M a d r i d ) 51B, 125 (1955). Kirrmann, ii., Federlin, P., Bieber, P., BUZZ.so?. chini. F m n c e 1954, 1466.
Luther, H., Lohrengel, E., Brennstof-Chem. 35, 338 (1951). >laminan, H. J., Sheppard, S . , J . O p t . SOC.Atner. 44, 815 (1954). Perkin-Elmer Instrument .Yews 5 , S o . 4 , 6 (1954).
REVIEW OF FUNDAMENTAL DEVELOPMENTS
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(18) Pershina, E. V.,Kaskin, S.S., DoiZady Akad. Nauk S . S . S. B . 100, 123 (1955). (19) Piaux, L., Gaudemar, Jl., Compt. rend. 240, 2328 (1U.5). (20) Puranik. P. G., J . Chem. P h y s . 23, 761 (1955). (21) Rice, B., Uchida, H. S., J . P h y s . Chem. 59, 650 (1958). (22) Robert, L., Spectrochim. Acta 6 , 115 (1953). (23) Rolfe, J. A . , Sheppard, D. E., Woodward, L. A., Trans. Faracday Soc. 50, 1275 (1954). (24) Rosenbaum. E. J.. Cerato. C. C . . Lauer. J. L.. J.Oat. SOL A m . 42, 670 (1952). ( 2 5 ) Shorygin, P. P., Kurina, L., Osityanskaya, L., M&&im. Aha 1955, 630. (26) Shull, E. R., J . Opt. SOC.A m e r . 45, 670 (1955). (27) Skinner, J., Spectrochim. Acta 6, 110 (1953). (28) Stamm, R. F.. A s a ~C. m x . 26, 49 (1954). (29) Takei, T., J . Chem. SOC.J a p a n . Pure Chem. Sect. 74, 965 (1953). (30) Welsh. H. L., Stansbury, E. J., Romanko, J., Feldrnbn, T., J . Opt. Soc. A m e r . 45, 338 (1955). (31) White, J. L7.< dlpert, N. L., DeBell, -4. G., Ibid., 45, 154 (1955). (32) Woodward, L. A . , Bill, P. T., J . Chern. SOC.1955, 1699.
M A X SWERDLOW, National Bureau o f Standards, Washington, D. C. A. 1. DALTON, National Institutes o f Health, Bethesda, Md. L. S. BIRKS, Naval Research Laboratory,Washington, D. C.
RENDS reported in the previous review (256) have continued to develop in a manner that has given electron microscop)- a responsible role in delineating the relationship of the microstructure of solids with their properties and behavior. This method in conjunction with other methods of analysis has not only established itself as a necessary line of research in chemistry, physics, and biology but is becoming a valuable aid in industry, :tgIiculture, and medicine. 1:ngineering developments in the transmission magnetic-type electron microscope have, in the past decade, made the instrument into a reliable laboratory tool. With most major countries inanufacturing a t least one electron microscope, recent advances iri electron optics should continue to be incorporated in the newer ( oniniercial designs. Nost current models are capable of a resolution of 20 A. (Radio Corp. of America, Camden, N.J., Type FXU-3B; Xorth Bmerican Phillips Co., Inc., Mt. Vernon, S.T.,Type EM-100A); one is capable of 6 A. (Siemens Br Halske .iktiengesellschaft, Werneraerk fur Messtechnik, Karlsruhe, Germany, Elmiskop I ) . Present knowledge about the inter:i( tion of electrons with matter indicates that a practical limit t o the resolving power of the electron microscope is about 5 A. Although this figure is two orders of magnitude worse than the theoretical limit imposed by the wave nature of electron beams, there is hope that the electron microscope may yet make visible the ultimate goal-the structure of the atomic lattice. Considerable effort still centers around problems related to optimum performance of the instrument. Approximately 1000 elertron niicroscopes are in use today. hIost are far from faultflee. Consistent high quality results are still matters for the well tiained specialist. The technical excellence of most of the electIon micrographs in evidence attests to their painstaking skill. Although high-resolution electron microscop)- is required for the elucidation of macromolecules, much of the experimentation is concerned with structural details just beyond the resolution of the light microscope. There has been a steady shift of emphasis
from problems related to instrumentation to problems related to specimen preparation. Mastery of these elements in electron microscopy is only prerequisite, however, to the more discriminating aspect of the problem-namely, the valid interpretation of the observed microstructure with regard to concepts concerning processes of growth, degradation, and function. The information and confidence gained from properly planned and controlled experiments combining the electron microscopical approach with other independent methods of analysis have resulted not only in confirming many present ideas but in revising and filling in gaps in existing knowledge.
MEETINGS AND SOCIETIES The scope and quality of meetings held by an increasing number of societies devoted to the science of electron microscopy provide good indications of recent developments. Annual conventions of national organizations were held in England, Germany, Japan, and the United States. Local regional meetings within the various countries are held with regularity to satisfy the wide interest in electron microscopy. The Annual Conference of the Electron Microscopy Group of the Institute of Physics was held a t Birkbeck College, University of London, on Xov. 10 and 11, 1953. As with previoue conferences of the British group, papers were read concerning the electron microscope and a wide range of its applications. Twenty-three papers were given reporting on electron optics, chemical applications, reflection microscopy, carbon, silica, and plastic replica techniques, thin sectioning, and particulate structures in biological materials. The proceedings were summarized by Challice (38). This same group held its 1955 conference on July 5 to 7 at the University of Glasgow. Forty-one papers were read: 16 on instrumentation and general techniques, four on crystals, plastics, and air pollution studies, and six on metallurgical applications.
598 The 6th meeting of the German Society for Electron Microscopy was held in Munster, Westphalia, March 28 to 31, 1955. Eighty-nine papers were presented. Six general survey papers from England, Germany, and Holland were read in the opening session. Later sessions included 13 papers on techniques of electron-optical apparatus, 11 on theoretical and experimental investigations of electron optics, 23 on general problems and preparation techniques, 16 on investigations in chemistry, metallurgy, and technology, 11on biological fine structure, and nineon virology and bacteriology. As in the past, Physik Verhand will probably publish abstracts and full reports wiII appear in Optilc and Zeitschrift jilr Wissenschaftliche Mikroskopie und fur Mikroskopzsche Technik. At the 12th annual meeting of the Electron Microscope Society of America held a t Highland Park, Ill., Oct. 14 to 16, 1954 (70), 82 papers were contributed, along Kith many invited discussions. Nine seesions were held, including instrumentation and specimen-preparation techniques, general biology and virology, and crystal growth in chemical and metallurgical applications. A number of special symposia were held on problems involved in the electron microscopy of viruses, electron metallography, electron diffraction, electron microscopy in petroleum technology, and industrial and applied microscopy. Because publication of many industrial investigations is restricted, symposia such as these provide a forum for exchange of ideas. The 13th annual meeting of the EMSA was held a t Pennsylvania State University, Oct. 27 to 29, 1955 ( 7 1 ) . There were only 37 contributed papers and three symposia, which gave more time for spirited discussion concerning interpretative aspects of the studies. I n this as in the 1954 meeting, the number of biological papers was not very much greater than those dealing Fith nonbiological materials. I n addition, there were symposia 6n electron optics, emission niirroscopy, and preservation and interpretation of structure in biological materials. There were no papers on work done with reflection electron microscopes and only three papers on x-ray shadow microscopes. The British seem to be doing most of the n-ork in both of these fields. A Symposium on X-Ray Microscopy and Microradiography sponsored by the International Union of Pure and Applied Physics has been announced for Aug., 16 to 21, 1956, a t the Cavendish Laboratory in Cambridge, England. The symposium will include all microscopical methods which utilize x-rays. Sessions are planned on the reflection, the contact, and the projection methods, and on applications in medicine and industry. The continued European interest in the theoretical and practical aspects of microscopy is further evidenced by the International Colloquium on Recent Techniques in klectron and Corpuscular (Particle-Ion) Microscopy held April 4 to 8, 1955, a t Toulouse, France. Twenty-six papers on high resolution, contrast, scattering, instrumentation, proton us. electrostatic microscopes, and related subject matter were presented by authorities from England, France, Germany, Holland, Japan, Sweden, and the United States. The proceedings are to be published in French. The conference was sponsored by the Minister of National Education, Centre National de la Recherche Scientifique, 13, Quai Anatole-France, Paris. The most significant meeting indicative of the growth and scope of electron microscopy ‘w’as the International Conference on Electron Microscopy held in London, England, July 15 to 21, 1954 (130). This meeting was sponsored by the Joint Commission on Electron Microscopy set up by the International Council of Scientific Unions with membership from the Unions of Physics, Chemistry, Biology, and Crystallography to provide a basis for the exchange of knowledge in this rapidly expanding field. The success of this conference can be measured by the large number of original research papers that were presented. Of the 163 papers that were offered, 46 came from Britain, 26 from Japan, 23 from France, 23 from Germany, 15 from the United States, eight from Holland, eight from Sweden, four from Switzerland, three
ANALYTICAL CHEMISTRY from Austria, three from Australia, two from India, one from Belgium, and one from Canada. Of these 70 dealt with biological subjects, 39 with instruments, 23 with metallurgical problems, 18 with electron optics, and 13 with industrial and chemical a p plications. Publication of the proceedings is in process (130). The success of this meeting demonstrated the need for periodic international gatherings and the groa ing need for permanent, continuing collaboration among the centers of electron microscopy. As a result, an International Federation of Electron Microscope Societies was formed in 1955. Biannual regional meetings are planned nrith an international conference every fourth year. The next regional meeting will be held in Stockholm, Sweden, on Sept. 17 to 19, 1956, and the 195Sinternational meeting is planned for Aachen, Germany. It is hoped that this international collaboration will bring about coordinated comniiinication in a subject that touches on almost all branches of pure and applied science. JOURNALS, BOOKS, AKD REVIEWS In January 1954 the Rockefeller Institute for Medical Research published the first number of the Journal of Biophysical and Biochemical C2/tology. This bimonthly publication is intended to provide a common medium for the presentation of morphological, biochemical, and biophysical studies of the structure of cells and their components and of the functions of these components. Investigations dealing with cellular organization at colloidal and molecular levels derived from the never approaches to cytology are favored. The editors will attempt to integrate information obtained from histochemistry, cytogenetics, cytochemistry, electron microscopy, and x-ray diffraction, The publication of “Precis d’0ptique Clectronique” under the direction of Pierre Grivet was announced by Bordas, Paris, France. Volume I, “Les Lentilles Electroniques” by P. Grivet, M-Y. Bernard, and A. Septier was due in April 1955. Volume 11, “Microscopes, Diffractographes, Spectographs de Masse et de Yitesse” by P. Grivet, M-Y. Bernard, E. Bertein, hl. Gauzit, and A. Septier was planned for the end of 1955. Volume 111, “Accelerateurs de Particules” by P. Grivet, ill-Y. Bernard, E. Bertein, J. Seiden, and A. Septier is to be expected early in 1956. Le Poole’s doctoral dissertation (147) constitutes a 93-page monograph of theoretical and practical significance in electron and ion optics. Chapter I describes how, by the application of magnetic lenses, the size of electron diffraction patterns can be made independent of the wave length associated with electron beams. Chapter I1 deals Kith the principles n hich have led to a simplified electron microscope. Using lenses of estremely short focal length, chromatic aberration is reduced by a factor of 5 compared to conventional microscopes. A resolving pon-er of 70 A. can be obtained even with high voltage fluctuations of 1%. Chapter I11 discusses the difficulties involved in obtaining accurate focus, means for getting sufficient accuracy, and a method for determining astigmatism in the objective lens. I n Chapter IV a mass spectrograph with two rotating electric fields is described. Haine (105) has contributed a critical review about the electron microscope. His informed evaluation and analysis of developments and problems concerning the instrument encompass 14 topics: resolving pori-er, image contrast, the magnetic objective lens, electrostatic lenses, possible methods for the correction of spherical aberration, astigmatism, the projector lenP, the electron gun, the object stage, alignment and focusing, vacuum, special features incorporated in some practical designs, and other forms and modifications of the electron microscope. Haine’s review concentrates mainly on the presentation and explanation of results rather than on the methods for their derivation. Although little mathematical theory is included, the chapter is most useful to the user and designer of electron microscopes. The 140 references deal with the instrument rather than its application. The main part is concerned with the magnetic transmission-type instrument, as this form is still the most important and
V O L U M E 28, NO. 4, A P R I L 1 9 5 6 most widely used. The present state of knowledge about the instrument is considered rather than a review of the stages in its development. On the other hand Hamm has written a chapter on electron microscopy (109) more concerned with the applications of t h e instrument. Preparation of specimens, the specimen-image relations, and the interpretation of electron micrographs of organic materials are described. Emphasis is placed on the interaction of electrons with the specimen and the evaluation of the factors that account for the formation and quality of the image. Alterations in the specimen leading to erroneous interpretations are discussed, together with techniques to avoid them. Application of the methods of electron microscopy to fibers, pigments, plastics, greases, soaps, crystal growth, and vapor condensates is well illustrated n i t h 25 micrographs. lrIany of these methods are also applicable to problems dealing with inorganic materials. This valuable chapter q i t h 84 selected references should assist chemists, physicists, and biologists in understanding and evaluating the present state of the a r t and the science of electron microscopy. Cosslett reviewed the most recent advances in lens design and commercially produced electron microscopes. The survey includes 51 references to instrumentation, specimen preparation, reflection microscopy, and applications in biology, metallurgy, and chemistry (43). Cosslett ( 4 2 ) has compared the practical limits of x-ray and electron microscopy. Nixon (173) has reviened the three forms of x-ray microscopes. His discussion, including 102 references, traces the developnient of the reflection, contact, and projection x-ray microscopes and considers the merits and limitations of each type. At present x-ray microscopes have attained a resolution of only 1000 A.-100 times lvorse than that of electron microscopes. The main advantages, however, are to be found in the penetration of thick, opaque metals and living biological specimens. The selected area x-ray diffraction in conibination a-ith x-ray microscopy offers the possibility of detailed identification of chemical compounds in areas 1 micron square. Some analysis of the chemical elements may be made from a consideration of specific x-ray absorption by the different parts of the specimen. Developments leading to a very fine focus electron beam generating x-rays from a small area on the target of an end rindow xray tube have allowed Cosslett and Pearson (45) and Nixon (172) to attain resolutions of the order of 1 micron on biological specimens.
BIBLIOGRAPHIES Because electron microscopy cuts across many of the classical divisions in science, its results are published in an ever-increasing number of articles, books, and periodicals. This dispersion and profusion of literature have become so vast that gaining ready access to this accumulated knowledge presents a real problem. Perhaps through the International Federation of Electron Microscope Societies a unified, improved means can be developed for disseminating knowledge of electron microscopy. I n the meantime compilation of current literature continues in various centers. Yon Borries and Ruska of the German Society for Electron Microscopy are continuing the publication of bibliographies in its official journal (247, 248). The Royal hlicroscopical Society (Tavistock Square, London, England) is still operating an abstracting scheme. The bibliography service offered by the New York Society of Electron hIicroscopists (2 East 63rd St., New York 21, N. Y.) is still available. Quarterly issues of current literature is published in the form of edge-notched cards (3.3 X 7.5 inches) punched according to both subject and author. About 1000 new cards were issued in the years 1954 and 1955. Since 1950 the compilation of recent literature in electron microscopy consists of more than 3600 references pertaining to electron microscopy, its techniques, instrumentation, and applications.
599 TECHNIQUES AND APPLICATIONS NONBIOLOGICAL
One of the most significant improvements in the technique of forming substrates and replicas has been the amorphous condensed carbon film developed by Bradley (32, 33). Evaporated films of carbon are very thin and very stable. They are resistant to electron beam bombardment and most chemical solvents. Replication processes are simplified and the final resolution can be better than 50 A. Valid interpretation of electron microscopical images and evaluation of the factors which limit resolution can be aided through an understanding of what happens when an electron beam interacts n-ith a specimen. Kanaya (133) obtained an analytical expression for the temperature distribution of a specimen on a thin substrate over a circular opening in the electron microscope as a function of the ratio of the radiation energy to that of conduction. TThen a conductive material such as gold is supported on a thin collodion substrate, its temperature is the same as that of the substrate and lower than that of the collodion alone. In the case of a protein specimen on a collodion substrate, the temperature of the specimen is higher than that of the substrate and also higher than that of collodion alone. No account was made of the accelerating potential and the amount of energy absorbed by the specimen. KO specific temperatures were measured. Astigmatism, and spherical, diffraction, and chromatic aberrations are four of the main reasons for image defects in electron optics. With 5 A. appearing as a practical limit to the conventional electron microscope, chromatic aberration caused by the interaction of a monochromatic beam of electrons with the substance of the specimen has a fundamental influence on resolution. hlarton, Leder, and Mendlowitz (164) have investigated this process from the standpoint of the energy distribution of electrons issuing from the average specimen. They find that a surprising number of electrons lose as much as 50 e.v. of their initial energy. When these inelastically scattered electrons equal the number of elastically scattered electrons, the worst condition arises, because the optimum image focus lies between the foci of two or more different velocities. The remedy for such aberration may lie in the use of filters which would cut off the low and high velocity electrons, or in the use of lens arrangements proposed by Le Poole (147). Transmission. In the past 2 years, the mo8t work on inorganic solids has been concerned with metals and alloys. A comprehensive review of electron metallography, specimen preparation, interpretation, and typical structural formations has been made by Habraken (102). Menter (159) has surveyed the uses and limitations of electron diffraction in the study of metals. Work on electron metallography of steel has been continued by American Society for Testing Materials Committee E-4, Subcommittee 11, with emphasis on interpretation of the bainite and martensite structures. Progress report 4 (6) of that committee concluded that the carbides in bainite and in tempered martensite were very similar in appearance. In both cases, the carbides tend to grow longer and parallel a t lower tempering temperatures (800” F.) and to spheroidize a t higher temperatures (llOOo F.), The committee has also extended its studies to nonferrous alloys with a titanium-manganese alloy under investigation but still in a preliminary stage. In addition to the committee reports, individual members of the ASTM group have made further interpretation of the first stages of tempering of martensite. Fisher (82) has used his “extraction replica” technique to show that the first stages of carbide formation consist of epsilon-carbide and FetC forms only after further tempering and a t higher temperatures. Austin and Schwartz (8) believe there may be other percarbides besides epsilon formed on initial tempering, based on careful electron diffraction correlation with electron metallography. Teague and Ross (240) have also observed initial tempering of
600 martensite with similar conclusions. I n addition Ross, Sernka, and Jominy (809)have studied steels of various compositions and concluded that the appearance of pearlite and bainite is largely independent of alloy composition. They also conclude the reactions of austenite-pearlite or upper or lower bainite all have the same form, in that ferrite plus carbides are the products in each instance, the only difference being in the mode of carbide precipitation. Examination and interpretation of minor phases in heatresistant alloys have been facilitated by double-etching techniques (%, 56). After the initial etch which brings out both small precipitates and minor phases, a second specific etch is used to attack what Brockway and Bigelow (35) term the y' phme, which is important in determining properties during high temperature aging. In this way y ' is determined, whereas it would otherwise be indistinguishable from carbides and nitrides. A emall amount of work has been done on the effect of stress on microetructure. Kehl and Bhattacharya ( 1 3 7 ) in a study of 1085 and 4340 steel reported no noticeable difference in the bainite strueture, whether the steels were under 60,000 pounds per aquare inch or no stress during isothermal transformation. On the other hand, Williams and O'Neill (258) examined several steela and stated that strain caused breakup and solution of boundary carbides. Substructure has been studied in copper by Pellier (nCe Delisle) (64),who showed that the substructure is different for single and polycrystalline specimens and changes after recrystallization on heating. She says that the size of the substructure blocks (IO00 A. for the smallest) is in reasonable agreement with theoretical predictions and some x-ray results. Hunter and Robinson ( I . a S ) have observed substructure in aluminum and its alloys; they state that the appearance is dependent on purity, orientation, and heat treatment, I n general, the long axis of the substructure blocks seemed to be normal to (100) crystallographic planes. Berghezan (18)has also studied substructure in aluminum alloys and claims that the striations are parallel to (111) crystallographic planes. Bussy (37) states that aluminum substructure appearance is different in different crystallographic planes. In evaporated films of aluminum-copper (239) oriented phases corresponding to Cu-Al2, Cu-B1, CurA14 were observed and checked by electron diffraction. Bismuth films (184) were found to recrystallize rapidly when irradiated by the electron beam. Gulbransen, McMillan and Andrew (101) have studied the oxidation of Armco and Puron in the 6.50' to 850" C. temperature range under pressures of 0.1 to 2 microns of oxygen. They used the oxide layers supported by Parlodion and found that the oxide crystals grow rapidly to a mosaic of large crystals. They also found that in iron containing an appreciable quantity of carbon, the oxide film reacted rapidly with the carbon, leaving a bright surface. Films formed on polished copper, nickel, and gold (162) by heating in vacuum (in hydrogen for the nickel) were found to be CuO, NiO, and pure gold. Cowley ( 4 6 ) has examined the oxide layer formed by heating copper in air a t 600" C. He shosTed that the intensity anomalies which differentiate the so-called CuO' electron diffraction pattern are not due to impurities, as has been suggested by Gulbransen and McMillan. The oxide grows in the form of long needlelike spines approximately perpendicular to the copper surface, with one or more screw dislocations along the axis of each spine. Each spine is a single crystal of CuO, about 1000 A. in diameter, elongated along the (110) zone axis. Intensity anomalies of the CuO' pattern may, therefore, be explained in terms of this particular morphology of normal CuO crystals resulting from growth about screw dislocations. Minute observations of the progress of change in a solid substance undergoing physical or chemical treatment is possible by means of electron microscopy combined with selected area electron dsraction. Sasaki and Ueda (213) have studied the alteration in a single crystal of Moo3 successively reduced with hydro-
ANALYTICAL CHEMISTRY gen for various periods of time ranging from 5 to 30 minutes a t temperatures ranging from 500' to 700" C. I n a certain stage of reduction atomic rearrangement can occur without affecting external shape. Thus the diffraction pattern can show profound changes without corresponding changes in the morphology ohserved in the microscope. Microdiffraction patterns of the reduced single crystal of MOO, showed some preferred orientation of AI0 and MOO, particles produced within the crystal. Similarly, Hashimoto (114) has shown that besides the knov 11 transformation of 11008 to 31002 resulting from electron beam bombardment in vacuum another type of reduction occurs: M O O S + ~1002' + MOO*
The intermediate phase was a monoclinic form differing from the end members. TTsing microscopy and microdiffraction, Suito and Uyeda (233, 234) related the striped extinction patterns and the lattice structure of 3-micron single crystals grown from gold sols. The thickness of the crystals varied from 30 to 120 A. These values \yere determined from the deviation of subsidiary diffraction spots from the conditions imposed by the Bragg equation, Hibi (116) and Yada (119) have studied imperfect crystals of metallic oxide smokes of molybdenum and magnesium, Unusual twins, elongations, and steps were observed in the crystal formations. The crystal habit (118) and the interference fringes association with magnesium oxide crystals ( 1 1 7 ) were further explained. The structural and morphological properties of sols and gels have always been of interest in chemistry. Watson, Parsons, S'allejo-Freire, and Souza Santos (249) have made an x-ray diffraction and electron microscopical analysis of the so-called Killstaetter's C-alpha, C-beta, and C-gamma gels of aluminum hydroxide, of special interest because of their adsorptive properties for enzymes and viruses. Different structures result, depending upon the method of preparation and the age of the gel. A sol and gel prepared by the same method, however, give identical x-ray diffraction patterns and the same morphologies in the electron microscope. Suito and Takiyama (232) have analyzed vanadium pentoxide sols made by two different methods. The sol prepared by cation exchange contained acicular crystals which did not grow upon aging. On the other hand, the so-called Blitz sol grew into orientated fibers upon aging. The relationship between color and the particle size of gold sols has been ascertained (160, 944). -4considerable literature has been established concerning the chemical composition and physical state of the compounds produced in the hydration of portland cement. Fundamental understanding of the process has been hampered by the lack of information regarding the size, shape, structure, and crystalline nature of the poorly defined gelatinous substances and other phases which make up the dynamic colloidal system during initial, intermediate, and final stages. Electron-optical methods in conjunction with x-ray diffraction, specific adsorption measurements, and chemical analysis have been recently applied to these problems (19, 26, 100, 236). Although the results of these investigations have been advanced mainly as preliminary or progress reports, present interpretations not only provide data on the morphology and crystalline state of the products of hydration but suggest the nature of the mechanism of hydration and the cementing action of portland cement. Monodisperse latices are important in investigating the mechanism of emulsion polymerization. They have also gained importance as secondary calibration standards in light and electron microscopy, light scattering and sedimentation studies, and aerosol studies. Bradford and Vanderhoff (31) have prepared monodisperse latices mith particle diameters ranging from 1000 to 12,000 A. Polystyrene, polyvinyltoluene, and various copolymers have been formed from carefully controlled emulsion
V O L U M E 28, NO. 4, A P R I L 1 9 5 6 yolynierizations. A series of polystyrene latices is available for distribution. The variations of particle diameter resulting from different substrates, electron irradiation, and other sources of eiior nere statisticalj. evaluated. Kienle and biaresh (158) have made observations on pigmented films. By means of replicas, thin sections, and studies of particle size, shape, and dispersion, they have provided a better understanding of the causes of and the changes in the surface reflectance of paints and coatings. Fling (141) has contributed to the understanding of the theory and niechanism of cleaning. By using a replica technique Kling and Mahl(142) have shown that the distribution of soil particles iii cotton and wool fibers is surprisingly uniform. Gotte, Kling, and Mahl (95) have shown that the number of dirt particles on the fiber surface is directly related to the lightness of shade of the textile. Coarser particles are more readily removed Particles smaller than 2000 A. become more difficult to remove, but migration of the dirt into the interior of cotton and wool fibers npparently does not take place. Rollins (207) has reviewed the use of light and electron microscopy in the study of the gross and fine structure of native cellulose fibers. Rollins and Tripp (208) have reported their studies of cotton fiber structure and Tripp, Moore, and Rollins (243) have studied the effects of some typical chemical environments on the isolated primary Fall of the cotton fiber. From these and structural considerations properties of interest to the textile technologist are discussed. Peck and Kaye (190, 192) have developed a technique for etching cellulose acetate fibers and films by direct solution of the surface molecules. A relatively undisturbed interior layer of polymer is uncovered for replication. Etching is achieved b j immersing the polymer for a short time in acetone a t -30" C. and then flooding nith an excess of cold absolute alcohol. Replicas reveal the orientation of molecules in the skin, the existence of voids, and pigment dispersions in cellulose acetate yarns. Modifications of this technique a ill undoubtedly aiise as new plastics are studied. Through the use of aluminumberyllium replicas, abraded surfaces and the skin of cellulose acetate fibers were studied. I t was found that ceramic guides for the fiber cause a sticking action, whereas metallic guides produce a shearing of the surface. The analysis aids in the quality control oi the production of synthetic fibers. By reflection electron microscopy Chapman and blenter (40) have studied directly the shape, surface structure, and frictional T\ e x of small fibers of Orlon, viscose rayon, cellulose acetate, glass I n the case of single fibers M 001, and undrawn and drawn nylon. of drawn n j lon, they found that under high sliding pressures the fiber undergoes considerably permanent deformation and severe tearing occurs. T o overcome the low beam intensity used to pievent damage to the specimen, a large angular aperture is needed in the objective lens. This and specimen motion reduce reaolution to about 1000 A. Although such resolution is only slightly better than with the light microscope, the great depth of field and the low viening angle permit a large part of the curved surface to be seen. By means of autoradiography and electron microscopy Hock (120) has obtained new information about the form and distribution of rosin sizing on a sheet of sized paper. His observations support the often expressed opinion-hitherto not adequately confirmed-that rosin size is not present as smooth surfacecovering films, but rather in the form of discrete particles about 1000 A. in diameter and that they occur either singly or in groups, insight into the structure and behavior of leather may be gained from studies by Swerdlow and Stromberg (237). The existence and niorphology of pores of the order of 150 A,, in radius, within collagen fibrils were delineated. Structural features of collagen from air-dried kangaroo tail tendon, impregnated ~ i t hmercury a t a pressure of 10,800 pounds per square inch, were compared with those of fibrils not exposed to mercury. A
601 helical configuration of subfibrillar elements was suggested by the electron micrographs of individual fibrils. The possible use of mercury under hydrostatic pressure as a technique in staining and preserving the structure of biological materials was advanced. These findings constitute a visual confirmation of the presence and probable size of small pores deduced from theoretical considerations of pore-size distributions in collagen and leather, and provide additional information about the shape, location, and arrangement of such pores in collagen fibrils. Anta1 and Weber ( 7 ) have reported an electron-optical study on the surface of glass. By depositing a thin film of bismuth on the surface of glass, they were able to overcome surface charging and obtain electron-diffraction patterns of the substrate-glasa -4calculation of the radial distribution function for a soda-limesilica glass surface indicated a deficiency of alkali atoms in the topmost atom layers, which is attributed primarily to water washing of the surface prior to examination. Terao and Sakata ( 2 4 1 ) have done some work on fresh fractures in glass, where the starting point of cracks was observed. It is claimed that artifacts due to reaction of the replica and the glass did not interfere with observation of the real structure. Direct electron microscopical evidence of structural inhomogeneities in glass has been reported by Prebus and Michener (198). Among several glasses investigated a grainy structure ranging from 20 A. up to a t least 200 A. has been observed. These have been interpreted as micellar regions which have a degree of order greater than that of the short-range order assigned to the glassy state by the random-network theory, but less than that of the long-range order characteristic of the crystalline state. Further study by the methods of electron optics and radiation scattering on glasses of known composition and thermal history is needed. An investigation of nucleation and crystallization phenomena at or near the liquidus region would lead to a better understanding of the process whereby a liquid changes into a crystalline mass. From such studies further insight into the glassy state of matter might be obtained. Thermionic Emission. Interest seems to be increasing in this type microscopy for studying metal surfaces directly a t elevated temperature. Rathenau and Baas (199) described instrumentation and activation of metal surfaces (such as iron-carbon alloys) with evaporated layers of barium, strontium, or cesium. They have shown examples of application to grain growth studies, interfacial angles and energy, and phase transformation. Septier (621) and blenter ( 1 5 7 ) have described applications to metallurgy, and hlenter has cautioned that the free surface examined by emission microscopy may not look the same aB the internal volume of the specimen-this is important in phase transformation. Heidenreich (115) has published a careful study of the elements which promote or poison emission from various metals; he finds carbon to be the most effective promoter for copper, nickel, and iron. He uses barium formate for activation and agrees with Baas that one should, if possible, use pure ironcarbon alloys rather than ordinary steel because the impurities poison emission below 700" C. He proposes a simplified mechanism for all the transformations from austenite; first + CY' )retained yrecr>stslll%edin n-hich the LY'is unstable and transfornis CY' + CY carbides Thus the same mechanism covers both high and low temperature transformation. Mollenstedt and Hubig (161) have discussed the film formed on metal surfaces by ion bombardment such as may exist in an emission microscope. Unless the metal is kept above 150" C., a structureless film will form on it in a partial vacuum (say 10-3 111111. of mercury); once formed this film is not removed by heating, and it obscures the structure one is trying to see. Reflection. Attention to reflection microscopy has increased the past few years, especially in England. I n general, the microscope is the same as for transmission, but the beam is inclined to strike the solid specimen a t grazing incidence (less the 10' angle); this results in higher magnification in one direction than in
+
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+
602
ANALYTICAL CHEMISTRY
the other. Menter (168) has shown applications to the study of cleavage surfaces on mica, growth steps, twinning, etc. Resolution of about 200 A. is claimed. Cosslett and Jones (44) described a variable incidence instrument in which electron diffraction could be accomplished without moving the specimen. Jones (131) developed a stage for heating the reflection specimen to 1000° C. while observing the image. As in other techniques, Fert, Marty, and Saporte (81) employ an electron beam with an illuminating angle of a few degrees, but the viewing angle is 25'. This reduces distortion and the extent of foreshortening of the enlarged image. With a narrow incident beam and a lightly etched specimen, surface relief of 20 A. is detectable, An extra gun for continuous ion bombardment is necessary to reduce surface contamination. A resolution of about 350 A. is claimed. Haine and Hirst (106) using illuminating and viewing angles which total 6" to 10" attain a resolution of about 430 A. A different approach in which the specimen acts as a "mirror" in a reflection electron-optical system has been used by Mayer (155) and by Bartz, Weissenberg, and Kiskott (16). I n this work the specimen surface is normal to the electron beam and what are actually observed are differences in potential on the surface. Surface irregularities as well as composition changes act as sources of potential difference, so the image is similar to the usual surface micrograph. A resolution of 3000 A. is claimed by 1Iayer (166), but he states that the theoretical limit should be a t least as good as with thermal emission electron microscopy, A new technique using the x-ray-sensitive properties of crystals or "rigid vinyl" plastic film allows relief images to be formed in x-ray microradiography (146). Replicas of these relief images can be observed in the electron microscope a t useful magnifications of 10,000 to 25,000 diameters. This technique is promising because it makes use of the penetrating power of x-rays and the resolution of the electron microscope. BIOLOGICAL
Technical advances have continued at a rapid pace during the past few years in practically all aspects of the technique for preparing biological material for examination n ith the electron microscope. The addition of low concentrations of silver nitrate to the drinking water of experimental animals for long periods results in the development of a vital stain as well as an electron stain of sorts, the silver, possibly in the form of a silver proteinate, being deposited in basement membranes of various glands, of renal glomeruli and proximal convoluted tubules, and of vascular endothelium. Intracellularly it was found in fixed and free machophages in various locations (69). W t h the exception of the choroid plexus, area postrema, (60, 246) neurohypophysis, and pineal body (60) in which the reaction is in general like that in other organs of the body (69), little or no silver was found to be deposited in the central nervous system (60, 246). These results do not necessarily imply that the penetration of ionic silver is correlated with silver deposition (246). Studies are a t present under way in several laboratoriee, the purposes of which are to develop new histochemical methods or adapt previously established histochemical techniques for use in electron microscopy. At this writing one article demonstrating the possibility of studying the sites of acid phosphate concentration (22.2) has appeared as the result of such studies. Another has demonstrated that methyl mercuric chloride reacts specifically with protein-bound sulfhydryl groups to produce electrondense areas in, for example, squamous epithelial cells involved in the process of keratin formation (11 ). The problem of fixation for electron microscopy remains one of primary importance. The veronal-acetate-buffered osmic acid fixative of Palade (182) remains the fixative of choice in the majority of laboratories and continues to give superior results. A chrome-osmic fixative (47) has been suggested as a useful adjunct to Palade's fixative and has been recommended as particu-
larly useful for studies on the central nervous system (155). A chrome-form01 fixative has been used for comparison with osmic acid to determine how much detail is visualized as the result of the deposition of osmium (151). There is a definite need a t the present time for a fixative not containing osmic acid, R hich will give equally faithful preservation of cell detail (51). One of the well recognized difficulties associated with the use of osmic acid is the determination of the degree of satisfactoriness of fixation. Because of the slowness of penetration it may frequently be observed in blocks of tissue fixed with osmic acid that of two immediately adjacent cells, one may appear well preserved and the other poorly preserved. I t is therefore necessary to consider this factor very carefully if the results of experimental intervention are to be interpreted correctly. I n the present situation it is essential to establish criteria for identifying good or bad fixation when either is present. Since osmic acid or fixatives containing osmic acid are the only generally acceptable fixatives for electron microscopy of tissue sections, the studies of Bahr (9, 10) on the reaction of osmic acid with various chemical constituents of protoplasm are of considerable importance. While little advance has been made in the area of fixation per se, there is some evidence that treatment after fixation may be of value in certain instances. Phosphotungstic acid has been used to increase the contrast of viral particles ( 107) and fibrous structures (218) and uranium nitrate has also been found to increase the contrast of certain virus particles (132, 238). All those who have carried out moderately extensive studies on the electron microscopy of tissue sections using methacrylate must have observed polymerization damage. Such damage may vary from extreme disruption in which even nuclear and cytoplasmic boundaries are unidentifiable, to minor effects such as occasional artificial interruptions in membranes bounding the nucleus, cytoplasm, mitochondria, etc. Polymerization damage, varying from block to block frequently makes interpretation of experimental intervention very difficult. Damage of this sort is less noticeable in tissue blocks, especially if the original blocks are small, but may be particularly severe in preparation of protozoa or cells of the peritoneal fluid, of blood, and of cells in tissue cultures. I t has been found, other things being equal, the substitution of benzoyl peroxide for Luperco (2,4-dichlorobenzoyl peroxide) will reduce polymerization damage (125). Prepolymerisation to a viscous state before introduction of the tissue will reduce polymerization damage under certain conditions (28). This practice is particularly valuable in obtaining final embeddings of the same consistency. Further, double embedding, first in 27, agar in 10% formalin immediately after fixation, followed by the usual dehydration and embedding in methacrylate, greatly decreases polymerization damage of free cells and of cells on free surfaces (78). I n addition, cells embedded in agar after fixation are handled as tissue blocks, eliminating the need for repeated centrifuging. Borysko (27) has demonstrated the value of high temperature embedding (60' to 80" C . ) in reducing polymerization damage in tissue culture and embryonic cells. Karrer (134) has demonstrated its value in reducing damage to cells on free surfaces (pulmonary alveolae and bronchioles). As for the embedding medium itself, various proportions of methyl and n-butyl methacrylate, from 1 to 4 (217) to 1 to 20 (94), are used to obtain the proper final consistence of the polymerized plastic. -4pparently the use of large-sized (00) gelatin capsules as containers results in hardening of the polymer to a degree which makes the addition of methyl methacrylate unnecessary. As far as thin sectioning is concerned, the glass knife continues to be the cutting edge of choice in the majority of laboratories, although the razor blade is still producing satisfactory results for Sjostrand (229). The possibilities of a polished diamond edge (80) are beginning to be explored (79). Modification of existing microtomes (56, 9 s ) and newly designed microtomes (195,227) are producing consistently satisfactory results. New additions to this group (80,225)appear to be equally satisfactory.
V O L U M E 2 8 , NO. 4, A P R I L 1 9 5 6
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Serial sections, while difficult to obtain, have been shown to be of value in determining the finer structure and interrelationship of cell coniponents (88, 164, 196). Athene screens (Sniethruet High-light, Ltd., Sidcot Heaton, Bolton, Lancs., England) of various designs are useful for this and other more general purposes. Sectioning artifacts-that is, loss of tissue by sectioning, modification of structure resulting from imperfection of the cutting edge and from compression during cutting-as well as artifacts induced by exposure to the electron beam have recently been empha-ized (167, 160). Knife marks originally evident a t low beam intensity may be modified or completely hidden by the flow of plastic occurring under high beam intensity. I t has also been demonstrated that substances that are incompletely denatured by fixatives such a5 formalin may be sublimed off and lost as a result of exposure to high beam intensity (169). Carbon supporting films have been found to eliminate specimen drift and to have about the same electron-scattering properties of the usual Formvar films. Such films specially prepared to possess small openings allow the examination of completely unsupported tissne for high resolution electron micrography (650). Undoubtedly the majority of investigators have experienced the difficulty of finding brush marks, glass chips, imperfections in the emulsion, etc., on their best negatives. Antiabrasive plates treated to resist exposure to high vacuum are available (Eastman Kodak Co., Rochester, S. Y.) as a-ell as fine-grained spectroscopic plates. I t nould seem reasonable to use plates of these types, even though greater expense is involved, since the photographic negative is the final result of all the preliminary effort. Several attachments which improve the efficiency of the RCA ERIU 2 series electron microscopes are available commercially. One of these is the externally centerable condenser lens aperture alloffing a choice of four aperture sizes. (Canal Industrial Corp., Washington, D. C,). Another is the deflection beam focuser, which greatly improves accuracy of focusing (Canal Industrial Corp.). A third is the below-focus objective aperture, of particular value because in this position there is less likelihood of contaniination (Canal Industrial Corp. and Illini Associates, Urbana, Ill.). Some of the results recently obtained by the application of these technical methods will now be considered in the folloa ing order: (1) virus studies, (2) bacterial morphology, (3) the structure of p l a i t cells, (4) general cytology of animal cells, and ( 5 ) special cytology of animal cells. Virus Studies. A good deal of the recent work in the virus field has been accurately and adequately revien-ed by Bang ( 1 2 ) . Only the pertinent papers which have come to attention since Bang's review are considered here. Before considering these articles, however, it will do 110 harm to re-emphasize the importance of utilizing the criteria described hy Bang for identification of a virus as those particles seen by electron microscopy: (1) characteristic appearance either as individual particles or as groups of particles, (2) association of the agent nith disease or infection, (3) physical testing of the association of particles with the infectiousness of the preparation, (4) association of particles nith other known activity of the virus, ( 5 ) agglutination of the particles by immune sera, preferably sera from convalescent animals, and (6) infection produced by a very few particles. It is important to bear these criteria in mind n-hen evaluating any claim regarding the identification of a particle seen in sections Aith the electron microscope as a particular virus. I t is generally agreed that when observed in thin sections, cells infected n ith virus contain particles which are elliptical or spherical in form and possess a central electron dense core and a single or double peripheral membrane separated from the core by a less dense region. The size of these particles varies from 500 to 3000 A. Cells infected with Herpes simplex virus contain primary bodies 300 to 400 A. in diameter and somewhat larger (400 t o 500 A . ) bodies covered by a peripheral membrane-both within the nucleus. Cytoplasmic particles n-ere larger and
603 possessed a double outer membrane 1000 to 1300 A. in diameter. These observations suggested that viral development is intranuclear and that upon migration to the cytoplasm the particles become mature (166). Vaccinia and fowl pox viruses on the other hand are associated with particles 2000 to 3000 A. in diameter originating in the cytoplasm. These particles possess a dense central body (nucleoid) and a single peripheral membrane. In the peripheral cytoplasm and extracellularly the nucleoid is larger and the peripheral membrane is double (166). Still another variant is the influenza virus. One study demonstrated that infected cells develop rods and spheres a t the cell surface, not within the cytoplasm proper or the nucleus (168). However, in another study ciliated cells of brochii experimentally infected with influenza were found to possess cytoplasmic inclusion bodies, which on examination with the electron microscope were found to consist of particles of the size estimated for influenza virus (110, 111). The intracellular form of particles associated nith infection with anopheles A virus has been described (85)and has been shown to develop in association with the membranes of the endoplasmic reticulum or ergastoplasm (84). I n addition, particles associated with the Shope fibroma have been classified in the same category a i t h particles seen in vaccinia and fowl pox (22, 149). Particles associated nith the Murray-Begg endothelioma were found both in an extracellular position and in the cytoplaFm. The latter particles showed continuity n ith membranes forming the wall of vesicles of undetermined origin (104). This latter relationship is possibly similar to that described in anopheles A infection (84)and to particles found ill the cytoplasm of the Cloudman melanoma S-91 (60). Particles again differing in size depending on whether they are intracytoplasmic (120 to 400 A.) or extracellular (1000 A.) have been found in mammary tumors of mice carrying the milk agent (21). I n cells of the Rous tumor extracellular and intracytoplasmic particles have been found to approximate the same size (500 A). The particles consisted of a thin peripheral membrane usually surrounding a dense central core (90). A representative of the psittacosis lymphogranuloma group, meningopneumonitis virus, has been studied, and the cells infected with it have been characterized as possessing particles of different size groups (2500 to 3000, 3000 to 4000 4000 to 5000, 5000 to 6000 A., and a still 9 larger form) exhibiting different internal structure. The presence of what appear to be budding forms suggests that these different particles represent different stages of a life cycle (91). \?rue like particles have also been found to be associated nith the Ehrlich mouse ascites tumor (217), with Sarcoma 37 (50) with spontaneous hepatomas of C3H mice ('i'r),and in the blood plasma of leukeniic mice (65). Particles 500 by 300 A. in a cryetallike pattern in the nuclei of cells infected nith an animal virus have also been described (140). A study on virus-host cell relationships hap shown that certain viruses will kill the host cells (RIengo and anopheles A), while others will multiply within the host cells but xi11 not destroy them (influenza and unadapted Newcastle virue) (83). .4 more general account and review of virus-host relationships have recently appeared ( I S ) . The special cases of insect polyhedral virus disease have been investigated and the macromolecular paracrystalline lattice and associated viral particles have been described (168). Stages in the development of a nuclear polyhedral virus disease have also been described. Elementary virus rods proliferate within the chromatin mass of the nucleus. They are then released into the nuclear sap. Membranous envelopes develop aroucd the rods and finally the polyhedral protein is deposited about the encapsulated viral particles (53). It has been suggested that the viral protein does not come directly from chromatin, although chromatin may be involved indirectly in the process of viral protein accumulation (261). Obviously, not in all of thePe investigations have the majority of the criteria ( 1 2 ) for establishing a definite causal relationship
604
ANALYTICAL CHEMISTRY
between a virus disease and a viral particle been applied, but the background of observations which is developing should soon make possible the clarification of life cycles in some instances and the classification of virus particles on the basis of niorphology in others. Before passing on to other areas an interesting and significant observation concerning bacteriophage should be recorded, The tail and tail fibers, not the head, are responsible for the primary state of absorption of phage to bacteria (259). Bacterial Morphology. Turning now to the morphology of bacteria-there have been claims in the past of the presence of formed elements of both cytoplasm and nucleus in these species. Recent evidence suggests that a nucleus of a sort containing deoxyribose nucleic acid does exist in bacteria. There is also some reason to feel that nucleoli exist. However, recent studies have produced no evidence of the presence of mitochondria in bacteria (89). The conservative view has also been expressed that inasmuch as what appear to be nucleoli may be cytoplasmic invaginations into a central vacuole and no membrane has been described bordering what has been called the nucleus, a strict homology with the nucleus and nucleolus of higher forms should not be made a t present (242). There is also evidence that tonicity of the fixative and the environment immediately before fixation are important factors affecting the finer structure of bacteria (206). I n any case a t the present time it is a relatively easy matter to distinguish bacteria and structures related to the presence oi bacteria in infected cells from all other structures present in uninfected cells (34). Investigations being carried out in several different laboratories should shortly clarify many aspects of the morphology of bacteria which are a t present disputed or unknown. This is an area in which studies of shadowed preparations of whole mounts will continue to give information not necessarily readily obtained from sectioned material (123, 144,145). Structure of Plant Cells. Primary emphasis in studies of the electron microscope of plant cells has been concerned with the structure of chloroplasts and grana, although spore walls (3) and walls of pollen grains ( 2 ) have been examined. Studies on yeast cells have also been made, in which it was found that ellsgrown on a glucose yeast extract were useful for studies on the cell wall and on bud and birth scars. owever, i t was necessary to grow cells on a presporulation me um in order to obtain material in which the detail of cell membrane, mitochondria, and nuclei could be observed in these sections (4). Studies on cahloroplasts have been carried out in Chlorella ( 5 ) , Chlamydoillonas (212), Aspidistra ( I @ ) , and Zea (122). The results of these studies indicate that chloroplast structure becomes increasingly complex with the appearance of grana in higher forms. Chloroplasts consist of stacked layers of membranes or laminae n i t h nhich chlorophyll is associated (122) Grana are more dense regions of chloroplasts in which the laminae become twice as thick as in the stromal portion of the chloroplast (231) and we approximately twice as numerous (122) Chloroplasts have heen described as developing from a small granule in young cells. This granule separates into a central dense core and a more peripheral stromal zone. Laminae develop as enveloping membranes around the dense core. Later additional laminae appear to be derived from previously formed ones. Mitochondria with characteristic internal structure have been described in plant cells ( 2 1 2 ) ,but further analysis and comparison of these as Tell as other cytoplasmic and nuclear components in relation to their counterparts in animal cells lie in the future. Cytology of Animal Cells. The large bulk of technically excellent high resolution electron microscopy of biological materials has been carried out on animal cells. This is true in part because a i t h this type of material satisfactory fixation, embedding, and sectioning were most readily obtained. I n the past few years the broad outline of the general cytology of animal cells has been filled in. The plasma membrane with its specializations and some of its activities, the fine structure of mitochondria,
@
the detailed structure of the Golgi complex, the small granules of the basophilic substance, the membranes of the endoplasmic reticulum, the nuclear membrane with its stomata, details of the nucleolus, nucleoplasm, and even chromosomes and the mitotic. figure have all been examined-in most cases with care and precision. I n the area of special cytology many differentiated cell types nith their particular characteristics have been competent1)analyzed. Specializations of the cell surface have been examined in a series of cell types. The striated border (256), brush border (202),and sterio-cilia of classical cytology have all been shown to consist of essentially similar microvilli. I n addition to the cell. long recognized as having a cuticular border, others such a? mesothelial cells (78, 176), cells of the gall bladder (263), and parietal cells of the stomach (214),embryonic yolk sac (55),and chorion (58) have been shown t o possess microvilli. hlicrovilli may vary considerably in length, in different cell types, but are in general characterized by having a constant diameter through most of their length and being relatively closely and regularly spaced. The characteristic structure of cilia (SO) has been extended and clarified. The nine peripheral paired filaments and t n o central filaments of cilia (76, 215) are also characteristic of the filaments of sperm tails (36). Cilia, of course, cannot be considered simply as a surface specialization of cells, since ciliary roots extend some distance into the cytoplasm and in the case oi sperm tails originate in relation to the centriole. However, the cell surface is involved in their development and formation and the preliminary observation ( 1 2 6 ) that microvilli sometimes possess the 9 and 2 arrangement of internal filaments has extremely interesting connotations. This would correlate well nith the finding of cilia and microvilli on the same cells of moluscan (76), amphibian ( 7 6 ) , and mammalian (76, 111) ciliated epithelium. Microvilli have been considered as of the same nature as the temporary cell processes of free cells (216); however, the latter are extremely variable in length, diameter, and spacing, and are frequently broadly based. Another difference is that in some instances, a t least, in proximal tubule cells of the kidney (202), in principal cells of the duodenum (5O), in epithelial cells of the gall bladder (263),and in mesothelial cells (78,184),coiled tubulai invaginations extend downward into the cytoplasm between the bases of the microvilli. Other examples of this correlation may come to light with more careful analysis of other cell types. These tubular invaginations may be comparable to vesicular structures associated with the plasma membrane of endothelial cells (179) and n i t h the vesicular component of Schwann and nerve satellite cells (62) R hich together on a finer scale may represent pinocytosis in these cell types. The basic process involved i n the formation of these invaginations is possibly the same as that involved in the process of phagocytosis, but when melanin granules approximately 1000 A. in diameter are injected into the peritoneal cavity, only macrophages phagocytose them ('78). Rheii thorotrast, a much-small-sized particle, is injected, both mesothelial cells and macrophages engulf them (176). This suggests the possibility that different physical-chemical phenomena may bp involved in the absorption of the two types of particles. Thus the problem of transport of material across plasma membranes is a complex one, involving solubility and semipermeabilit) , pinocytosis and phagocytosis. The problem of the movement of materials in solution across semipermeable membranes can probably be better approached by means other than the electron microscope. As indicated above, hov ever, the electron microscope is useful in studying the mechanisms involved in pinocytosis and phagocytosis and should eventually give some evidence as to whether they are one and the same. This n-hole question is further complicated by the suggestion that the plasma membranes invaginated during the process of pinocytosis become part of and continuous with the membranes of the endoplasmic reticulum or ergastoplasm (181, 254). These membranes in turn are in con-
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V O L U M E 28, NO. 4, A P R I L 1 9 5 6 tinuity n ith the nuclear membrane ( 2 5 1 ) and apparently in some instances with membranes of the Golgi complex (187). This euggestion of the origin of intracellular membrane systems is an intriguing one [the evidence for their intracytoplasmic origin is equally good ( 2 5 4 ) ] ,but a corollary to this hypothesis must be an explanation of how cells such as ameba and macrophages decide which plasma membrane to incorporate into cytoplasmic membrane systems and which to discard. Evidence from timelapse photomirrography shom ing rapid and continuous incorporation of vacuoles a t the cell surface certainly indicates that, if all invaginated plasma membranes were retained in the cytoplasm, macrophages would soon consfst of nothing else. .Lictually the evidence for this relationship is at the present time mostly circumstantial. During the past few years the details of the structure and relationships of cytoplasmic membrane systems have been clarified somewhat. The basophilic substance of cytoplasm (ergastoplasm) in its most highly specialized form has been shown to consist of a system of double membranes which are arranged in parallel rows or concentric circles (228). I n this condition some anastomosis between rows of membranes occurs and it has been shown that these membranes are actually profiles of flattened saw (254). I n addition, these membranes have been demonstrated to be the cut profiles of membranes of the endoplasmic reticulum (187). These membranes with their associated small granules (180) vary greatly in number and complexity from one cell type to another (184). There is clear evidence now that cytoplasmic basophilia is related to the small granule component (23, 180, 194). It has also been suggested that they are the primary site of concentration of cytoplasmic ribose nucleic acid (185), although there is evidence to indicate that large amounts of ribose nucleic acid are present elsewhere in the cytoplasm (143). The granules may be closely associated with a highly developed membrane system as in exocrine cells of the pancreas (228) and cells of the parotid gland (184),partly free and partly associated with membranes as in principal cells of the duodenum (19$), tumor cells (50),and nerve cells (187). The establishment of an acceptable terminology which can be used to describe all variants and combinations of these two components, the small granules of Palade, and the membrane system is necessary but not easy. The small granules are refiponsible for the basophilia and the membrane system for the apparent filamentous or lamellated appearance originally described for ergastoplasm. The term “ergastoplasm” would be historically accurate and descriptively correct when applied to heavily basophilic cells such as those of the pancreas and parotid gland, but its use in describing the same components in strongly acidophilic cells such as parietal cells of the gastric mucosa would require some modification or extension of the original meaning. A similar or greater difficulty is encountered in attempting to apply the phrase “endoplasmic reticulum” to all cell types. Obviously, when the term was first coined (196),it applied to both the membrane system and the small granules associated with the membranes. I n addition, there are many instances in which the system is not limited to the endoplasm of the cell and in other instances the membranes do not form a continuous reticulum. It is uncertain as to M hether in the near future terminology can be standardized for these cytoplasmic components which will be scientifically accurate and historically correct, and mill a t the same time satisfy national and personal loyalties. What is certain, however, is that when the term “ergastoplasm” is used, it is used in reference to the combination of Palade granules and the membrane system ( 2 3 ) and that the term “endoplasmic reticulum” refers only to the membrane system itself (184). The oiigin of small Palade granules is a t present under investigation and discussion. The possibility exists that they originate de nouo in the cytoplasm, although recent racer studies suggest that they originate in the nucleus and pass into the cytoplasm later. This question of origin and also the related one of
605
continuity between nucleoplasm and c) toplasm may have been answered in recent studies on the nuclear membrane, The earlier observation that the nuclear membrane is a double structure has been confirmed in one sense and modified in another. There is no question but that two membranes exist; however, it has recently become clear that the outer of these two membranes is the inner boundary of the cytoplasm and that as such it is iii continuity with the membranes of the ergastoplasm or endoplasmic reticulum (60, 180). I t also seems certain that the two membranes become continuous at the margin of what appear to be points of continuity between nucleoplasm and cytoplasm (50, 180, 251). However, a thin membrane has been seen extending across these openings (1) and densitometric analysis of electron micrographs indicates an increase in osmiophilia a t exactly the area where the openings should be (155). The numerous perforations described in some nuclear membranes, notably paramecium (251), may be suspect because of the possibility of contraction during the slow penetration of osmic acid into large cells covered by a dense pellicle. However, there would appear to be no question but what under certain experimental condition* definite modification in the structure of the nuclear membrane may take place (112). Unquestionably in such a dynamic system as the animal cell, some means of transport between nucleus and cytoplasm must exist, but whether these so-called nuclear pores subserve this function remains to be proved. The fine structure of the Golgi complex was recently described in cells of the epididymis as consisting of a system of smooth double membranes, small granules or vesicles, and large vacuoles. The three components were clearly shown to be in exactly the same area as the classical reticulum in osmic impregnated cells (49). Since this demonstration the three basic components have been described in good detail in duodenal cells (51, 6 2 ) , pltncreap (51, 62, 229), plasma cells (23, 51, 52), hepatic cells (62, 75, 103), white blood cells (24,192), spermatids (36,61, 75,96), cells of the hypophysis (73, 74, 103) and thyroid ( 5 7 ) , and nerve cells (61, 187). I n tumor cells the complex may be highly developed (103) or extremely small, consisting only of a few membranes and vesicles (60, 128). It has been suggested that because the claim of artifact has been made against the classical Golgi apparatus, because many different things have been included under the name Golgi substance, because of the variable morphology of the structures concerned, because of uncertainty as to whether this fine structure actually is the electron microscopical representation of the classical Golgi reticulum, and because of the evidence of continuity between membranes of the Golgi complex and those of the ergastoplasm or endoplasmic reticulum, a new term “agranular reticulum” should be substituted (187). I n spite of the above objections the term Golgi complex or apparatus should be retained for several reasons. First, the label of artifact applies in no greater degree to the fine structure of this cell component than to any other. Secondly, with the resolutions obtainable n-ith the electron microscope there is no difficulty distinguishing the Golgi complex, whether highly or poorly developed, from an! other cell structures and thus there is no longer any reason for confusion. (We continue to refer to mitochondria, even through a t one time they were frequently confused with bacteria, secretory granules, and melanin granules.) Thirdly, the contents of the small vesicles, large vacuoles, and the area enclosed within the membrane systems of the Golgi complex reduce osmic acid R hen no other components of the cell do, indicating a distinct difference between the Golgi complex and ergastoplasm ( 4 8 ) Fourthly the term “agranular reticulum” is inappropriate because it fails to include reference to the small vesicles and large vacuoles, which are clearly separate although undoubtedly derived from the membrane system of the complex. The characteristic finer structure of mitochondria, once described (183),has been extended to include the mitochondria of variety of cell types. Differences in detail have been found (61, 197, 202, 216, 228, 252); however, with but few exceptions, the
606 presence of a continuous outer peripheral membrane and an inner peripheral membrane extending as cristae in the form of flattended sacs or villi into the mitochondrial matrix has been confirmed. The suggestion that a continuous peripheral membrane is sometimes lacking (197, 252) may probably be discounted on the basis of less than adequate preservation or resolution, The suggestion that mitochondria may be involved in the intracellular concentrations of cations (255, 257) is an interesting one and should stimulate investigations aimed a t verifying and extending it. The general area of the relationship between fine structure, function, and biochemistry of mitochondria is so large that it would require a separate review of its own. In this area the great value of electron microscopy will be as a service to biochemists in determining the degree of intactness of isolated mitochondria. One other special function suggested for mitochondria is their direct involvement in the formation of secretory granules in pancreatic cells (39, 86). This suggestion brings up the general question of the relationship between secretory activity and the various cytoplasmic components. It has been indicated that under certain conditions ergastoplasmic sacs are directly involved in the formation of secretory granules in pancreatic cells (254). It has also been suggested that the small vesicles of the Golgi complex enlarge to form secretory granules (229). Electron micrographs may be produced to give evidence for any one of these three hypotheses. On the other hand, there is clear evidence that the Golgi complex is directly involved in the formation of the acrosome in spermatids (36),in the formation of specific granules in cells of the anterior hypophysis ( I O S ) , and in the absorption and modification of and release of lipide material from principal cells of the duodenum (856). It would appear that the hypothesis which most satisfactorily takes into account all of the currently available evidence is that the Golgi complex is primarily involved in the removal of water from maturing secretory substances, on the one hand, and engulfed or absorbed material on the other (51, 86, 87). Some information has recently become available regarding the fine structure of nucleoli, nucleoplasm, chromosomes, and the achromatic spindle of mitotic figures. Nucleoli, whether of normal or malignant cells, are composed primarily of a concentration of small granules similar in size and electron density to the small granules of Palade in the cytoplasm. Interspersed between these concentrations in a spongelike arrangement are less electrondense areas containing both small granules and fine anastomosing filaments (20, 50). Occasionally associated with nucleoli are large areas free of granules but containing numerous anastomosing filaments approximately 70 A. in diameter. These areas may represent nucleolar associated chromatin (50). Chromatin bodies of the macronucleus of a protozoan have been shown to vary from a dense homogeneous sponge work in adult organisms to highly organized structures (appearing as a honeycomb in cross section and as parallel lines in longitudinal section) in old individuals (210). hfeiotic chromosomes shorn a variation in granule and filament content, the filaments differing in diameter depending upon the stage of division. Very fine (28 f 7 A , ) threads are thought to represent single deoxyribonucleoprotein molecules (66). Thin tubular elements not encountered in resting cells are thought to represent elements of the achromatic spindle (193). I n some meiotic chromosomes, in addition to the granular and filamentous component, a central compound core of tubules has been visualized (170). Serial sections suggest the possibility that nuclear blebs containing chromatin and nucleolar material may be separated off to be distributed in the cytoplasm (89). These are what are considered to be the significant observations on the internal structure of the nucleus a t the present writing. The increased resolution resulting from thinner sections and generally improved technique have made these preliminary observations possible and more complete studies on nuclear detail and the interrelationships of nuclear components may be forthcoming in the near future.
ANALYTICAL CHEMISTRY Special Cytology of Animal Cells. The contributions of electron microscopy to special cytology-that is, to our knowledge of the finer structure which characterizes the cells of various tissues and organs-have been very numerous in the past few years. They have been so numerous in fact that an attempt is made here only to list the cell types involved, to make short comments on their special features, and to record the references to the articles in which further detail will be found. Recent studies on the intercellular matrix have served to confirm earlier studies on the structure of collagen and elastin (800) and have demonstrated the existence of a monomeric form of collagen (tropocollagen), which is probably the precursor of periodic mature collagen in physiological fibrogenesis (98, 99). Interestingly enough, these studies have not brought to light anything like what might have been expected from silver impregnation and light microscopy in the way of intercellular cement. The terminal bar apparatus of light microscopy appears to be more a thickening of plasm membrane than a concentration of extracellular material (263). The various types of blood cells have been examined and their specific granules described. They have been shown to possess typical mitochondria, moderate amounts of ergastoplasm, and a well developed Golgi complex (24, 97, 136, 229). Plasma cells have been shown to possess large amounts of highly organized ergastoplasm and a relatively hypertrophied Golgi complex (24, 51, 52). The formation of platelets from megakacyocytes has been described (268). A beginning has been made on the fine structure of peritoneal fluid cells and the characteristics of mesothelial cells from several sites have been noted (78, 175). The structure of endothelial cells (179), the lining of sinusoids of the pituitary gland (203), and the wall of the aorta have been analyzed (188). The fine structure of cardiac and skeletal muscle has received special attention and as a result nun:erous papers concerned with these subjects have appeared recently. In a review of the general field of the structure of striated muscle through part of 1954 ( 17 ) , it has been indicated that the basic structural organization of myofibrils, myofilaments, cross filaments, cross banding, sarcoplasmic reticulum, sarcosomal fine structure, and myo-tendinal junction are similar in cardiac and skeletal muscle, whether of vertebrate or arthropod. Still more recently further details of the structure of skeletal muscle with some consideration of the relationship between structure and function have been supplied (211). I n addition, a greater number and concentration of mitochondria in red muscle than in white muscle of insects have been noted (68). A recent paper contains excellent detail of the fine structure of insect flight muscle, with an interpretation of this fine structure in relation to the known chemistry of muscle contraction (121). I n vertebrates, mitochondria are more numerous in cardiac than in skeletal muscle and in some instances appear to possess tubular rather than the flattened form of cristae (253). It has also been noted that mitochondria are as numerous in insect flight muscle as in vertebrate cardiac muscle (139). One distinguishing feature of cardiac muscle, the intercalated disks, has been investigated, and it has been found that myofibrils are not continuous through it. Thus intercalated disks actually represent cell boundaries in cardiac muscle (226, 245). I n the study of nervous tissue considerable attention has been given to the synaptic fibers. The presynaptic fiber can be identified by the presence within it near the synapse of numerous vesicles 200 to 500 A. in diameter (63,64). These vesicles and mitochondria were found to be much more numerous on the presynaptic side of the synapse than on the dendrite side and a similar concentration of vesicles was a t the axon endings of neuromuscular synapses (178). Degeneration, first of the synaptic vesicles, followed by lysis of mitochondria was found to occur in presynaptic fibers of the ventral acoustic ganglion after destruction of the cochlea (61). I n the crayfish giant synapse on the other hand, vesicles appeared to be concentrated on the postsynaptic
V O L U M E 28, NO. 4, A P R I L 1956 side (20.4). Numerous vesicles and mitochondria are also characteristic of receptive nerve endings in association with the hair cells of the cochlea (69). The method of formation of the myelin sheath of vertebrates has been clearly demonstrated in embryonic material. Nerve fibers become invaginated into the accompanying Schwann cell cytoplasm, the plasma membranes of both nerve fiber and Schwann cell remaining intact. Rotation of the Schwann cell around the nerve fiber results in the building up of numerous helical layers of membranes in which the lipoproteins of the myelin sheath are laid down (94). A similar relationship has been demonstrated for invertebrate material (92) and this basic organization has been demonstrated in adult vertebrate nerve fibers (205). A definitive paper on the fine structure of the nerve cell body has recently appeared in which the details of the Sisel substance have been shown to be comparable in a general way to the ergastoplasm or combination of endoplasmic reticulum and Palade granules in other strongly basophilic cell types. I n addition, excellent examples of the Golgi complex of neurones (called in this paper “agranular reticulum”) were described. Fine fibrils 60 to 100 A. in diameter coursing through the cytoplasm of the neurones were identified as neurofilaments (187). A monograph on the fine structure of the thalamus has appeared ( 7 9 ) . Preliminary notes on the electron microscopy of neuroglia ( 7 2 ) of the spinal cord ( 1 5 2 ) , cerebral cortex (113, 156), neurohypophysis (67,186), and the organ of Corti (230)indicate that shortly a good deal more information on the fine structure and interrelationships of components of the nervous system Till be made available. A survey of recent observations on the fine structure characteristic of cells of certain organs reveals that valuable new information has been made available about the cells and cellular relationships in the skin, lung, stomach, intestine, pancreas, liver, kidney, and testis. A thin basement membrane at the dermoepidermal junction has been clearly visualized (177, 218). Bundles of tonofilaments less than 100 A. in diameter have been homologized R ith the Herxheimer fibers of light microscopy. Similar filaments embedded in opposing elongate granules of adjacent cells make up the intercellular bridges. Filaments do not cross the narrow space intervening b e t w e n the granules (219). A preliminary report on the more superficial layer of stratified squamous epitlieliiini has also appeared (218). I n the lung, continuity of an epithelial lining of the alveolae has been clearly established (160) and i t has been shown that the thickness of the cytoplasm of endothelial cells of pulmonary capillaries may be as thin as 100 A. (154). I n parietal cells of the stomach (214) and principal cells of the intestine (266j evidence has b e m presented correlating fine structure with absorptive processes. I n the exocrine cells of the pancreas surprisingly little change in intracellular components was noted following injection of pilocarpine (229). I n this cell the distinction between the ergostoplasm and Golgi complex is particularly clear (48). Further studies on the relationship between secretory activity and modification of fine structure in this cell type are clearly indicated. A definitive paper on the fine structure of hepatic cells ( 7 5 ) showed that as a result of feeding after a fast, new membranes of the ergastoplasm appear first near the periphery of the cell, the small granules, probably derived from the nucleus, being added later. The mortise-tenon-like interdigitation of the lateral borders of hepatic cells and the villiform processes of the cell surface facing the sinusoid were also described and evidence for an incomplete sinusoidal lining was presented. I n the kidney the glomerulus has received considerable attention primarily in regard to its normal structure (14 , 108, 171, 189, 201, 264) but also in regard t o pathologic changes ( 2 2 4 ) . The concept of the basic organization of the glomerulus (174) has not been greatly modified by these more recent studies. Higher resolution has resulted in a clearer understanding of the structure of the basement membrane (201, 264) and i t has been
607 suggested that the perforations of the endothelial lining are not true openings but are covered by a very thin film of endothelial cytoplasm (864). High resolution electron micrographs have given us further information concerning the fine structure of the proximal tubule cell under normal and experimental conditions (202) and of the cells of the thin segment, distal, convoluted tubule, and collecting tubule ( 189). An interesting observation, aside from vertebrate kidney cell structure, is the peculiar orientation of mitochondria in cells of the Malpighian tubule of the grasshopper (16). The groundwork has thus been laid for further experimental work and an understanding of pathologic changes iii kidney cell ultrastructure. As far as the testis is concerned, of primary interest is the description of the differentiation of spermatids in the cat (36). I n this study the intimate relationship of the Golgi complex to the formation of the acrosome is demonstrated as well as the detailb of structure and relationships of the centriols and mitochondria t o the development of the tail filament and sheath. Khile still furthei work of a purely descriptive nature is necessary in many areas, i t is clear that the basis has been laid in many other areas for experimental analysis. Descriptive studies of high quality have made i t possible to predict that an increasingly high proportion of the publications in the general area of electron microscopy of tissue sections will involve the experimental approach. They have also made possible a comparison between normal and malignant cells, the beginning of which has already been made (41, 50, 127, 128, B O ) , with some assurance that any quantitative or qualitative differences uncovered would be valid.
ACKNOWLEDGMEKT Many contributors to this field were generous and helpful in making available reprints, abstracts, and references t o their papers and talks. Their cooperation is sincerely appreciated. LITERATURE CITED (1) Afzelius, B. AI., Ezptl. Cell Research 8 , 147 (1955). (2) Afzelius, B. hI., Erdtman, C. T., Bot. Notiser 108, 138 (1955). (3) Afzelius, B. hI., Erdtman, C. T., Sjostrand, F. S., Scensk Botan. Tidskr. 48, 155 (1054). (4) Agar, H. D., Douglas, H. C., J . A p p l . Phys. 26, 1393 (1955). (5) Albertsson, P. A,, Leyon, H., Exptl. Cell Research 7, 288 (1954). (6) Am. S O C .Testing Materials, Proc. 54, 568 (1954). (7) Antal, J. J., Weber, A. H., A c t a Cryst. 7, 122 (1954). (8) Austin, 8. E., Schwartz, C. AI., ASTM, 1916 Race St., Philadelphia, 13, Pa., 58th Annual Meeting, Atlantic City, 1955. (9) Bahr, G . F.. Exptl. Cell Research 7, 457 (1954). (10) Ibid., 9, 2 i 7 (1955). (11) Bahr, G. F., RIoberger, G., Ibid., 6, 506 (1954). (12) Bang, F. B., Ann. Rev. Xicrobiol. 9 , 21 (1955). (13) Bang, F. B., Federation Proc. 14,619 (1955). (14) Bargmann, IT., Knoop, A., Schiebler, T. H., Z . Zellforsch. u. niikroskop. A n a t . 42, 386 (1955). (15) Bartz, G., Weissenberg, G., Wiskott, D., Proc. Intern. Coflf. Electron Microscopy, London, 1954, Royal Microscopical Societv. Tavistock Sauare. London. in Dress. (16) Beams, H . W., Tahmisian, T. N., Devine, R. L., J. B w p h y s . Biochem. Cytol. 1 , 197 (1955). (17) Bennett, H. S., Bm. J . Phys. M e d . 34, 46 (1955). (18) Berghesan, A., Publ. Sci. & Tech. du hlinistQre de l’Air, S o . 283 (1953). (19) Bernal, J. D., “Proceedings of Third International Symposium on Chemistry of Cements, London, 1952,” Paper 9, Cement and Concrete Assoc., London, 1954; Reinhold, Kew York, 1954. (20) Bernhard, W., Bauer, A., Gropp, A., Haguenau, F., Oberling, C., Erptl. Cell Research 9, 88 (1955). (21) Bernhard, W., Bauer, A., Guerin, bl., Oberling, C., Bull. cancer 42, 163 (1955). (22) Bernhard, W., Bauer. A., Harel, J., Oberling, C., Ibid., 41, 423 (1955). (23) Bernhard, W., Gantier, A., Rouiller, C., Arch. A n a t . M i c r . et M o r p h . E z p t l . 43,236 (1954). (24) Bernhard, W., Haguenau, F., Leplus, R., Rev. hBmat. 10, 267 (1955). (25) Bigelow, W. C., Amy, J. A., ASTM 58th Annual Meeting, Atlantic City, 1955.
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