Microscopy Microanalysis Microsc. Microanal. 9, 96–138, 2003 DOI: 10.1017/S1431927603030113 AND © MICROSCOPY SOCIETY OF AMERICA 2003 Key Events in the History of Electron Microscopy F. Haguenau,1 P.W. Hawkes,2, * J.L. Hutchison,3 B. Satiat–Jeunemaître,4 G.T. Simon,5 and D.B. Williams 6 1 Laboratoire de Médecine Expérimentale, Collège de France, F-75005, Paris, France CEMES-CNRS, B.P. 4347, F-31055 Toulouse cedex 4, France 3 Oxford University Department of Materials, Parks Road, Oxford GB-OX1 3PH, UK 4 Institut des Sciences du Végétal, UPR 2355 du CNRS, F-91198 Gif-sur-Yvette, France 5 25, Park Lane, Ancaster, ON L9G 1K9, Canada 6 Department of Materials Science and Engineering, Lehigh University, Bethlehem, PA 18015-3195, USA 2 1. B ACKGR OUND It is not easy to understand how the electron microscopes and electron microscope techniques that we know today developed from the primitive ideas of the first microscopists of the 1930s. Newcomers to the subject in particular, their time almost fully occupied with grasping practical methods and modern computing techniques, can rarely devote much attention to the history of their subject. For some, however, this is a source of frustration: If a guide to the principal stages in the development of the subject and to the main actors and their publications were available, they would find the time to study it. Some years ago, Gérard Simon suggested that such a guide would be appreciated; shortly after, Simon’s health prevented him from pursuing the project and the task of collecting suitable references was taken over by Françoise Haguenau and Béatrice Satiat–Jeunemaître ~life sciences!, David Williams and John Hutchison ~physical sciences!, and Peter Hawkes ~electron optics and instrumentation!. We have appealed to numerous colleagues for suggestions, as Received September 10, 2001; accepted September 19, 2002. *Corresponding author. E-mail: [email protected] many as possible of which have been incorporated in the following list. Nevertheless, this remains a personal choice—we have no doubt that equally deserving papers have been omitted and suspect that some undeserving ones have been included. The coverage is essentially limited to the traditional forms of transmission and scanning electron microscopy. Some specialized modes of operation are absent, notably reflection electron microscopy and many aspects of surface microscopy. 2. E LECTR ON O PTICS I NSTRUMENTATION AND 1897 Announcement by J.J. Thomson ~Cambridge! that cathode rays consist of exceptionally light ~or extremely highly charged! particles; the fact that these were indeed exceptionally light was confirmed in 1899 and the particles soon came to be called “electrons.” Thomson, J.J., Cathode rays. Electrician 39 ~1897! 104–109; Phil. Mag. 44 ~1897! 293–316. 1923 Louis de Broglie ~Paris! introduces the notion of a frequency, and hence a wavelength, for particles, History of Electron Microscopy l 5 h/mv. Several notes in C.R. Acad Sci. Paris culminate in the full account. Broglie, L. de, Recherches sur la théorie des quanta. Ann. Physique (Paris) 3 ~1925! 22–128, reprinted in Ann. Fond. Louis de Broglie 17 ~1992! 1–109. 1926–1927 Hans Busch ~Jena! shows that the focusing effect of a rotationally symmetric magnetic field can be described by the lens formula. Busch, H., Über die Wirkungsweise der Konzentrierungsspule bei der Braunschen Röhre. Arch. Elektrotech. 18 ~1927! 583–594. 1927 George Paget Thomson ~Cambridge! and Clinton J. Davisson & Lester H. Germer ~Bell Telephone Laboratories, New York! demonstrate electron diffraction. Thomson, G.P., Diffraction of cathode rays by thin films of platinum. Nature 120 ~1927! 802. Davisson, C.J. and Germer, L.H., Diffraction of electrons by a crystal of nickel. Phys. Rev. 30 ~1927! 705–740. 1928 Beginning of work on electron lenses in the Technische Hochschule Berlin ~Laboratory of Adolph Matthias!; under Max Knoll, Ernst Ruska begins research that leads to the first two-stage electron microscope ~1931!. The first image had a magnification of 316. Knoll, M. and Ruska, E., Das Elektronenmikroskop. Z. Physik 78 ~1932! 318–339; Beitrag zur geometrischen Elektronenoptik, I, II. Ann. Physik (Leipzig) 12 ~1932! 607–640 and 641–661. 1931 Ernst Ruska shows that Busch’s lens formula is correct. Ruska, E. and Knoll, M., Die magnetische Sammelspule für schnelle Elektronenstrahlen. Z. Tech. Phys. 12 ~1931! 389–400 and 448. 1931 The Rüdenberg patent episode; the first patent for an electron microscope was applied for on May 30, 1931 by Siemens–Schuckert Werke ~Berlin!, Rüdenberg’s employer, and the patent was later recognized as Rüdenberg’s in the United States. There is, however, no suggestion that Reinhold Rüdenberg ever constructed a microscope and it is generally believed that he applied for the patent as a result of a report by Max Steenbeck, who visited the Technische Hochschule several weeks before the date of the patent 97 application. For an exchange of letters between Knoll and Steenbeck concerning this point, see The Beginnings of Electron Microscopy ~Adv. Electron. Electron Phys. Supplement 16, 1985, 602–608!. 1932 Walter Glaser begins theoretical studies on electron optics. His work is paramount for the future development of the subject ~see 1952!. 1933 Construction by E. Ruska of a two-stage electron microscope with three magnetic lenses, condenser, objective, and projector. Images of cotton fibers and aluminum foils were obtained. A magnification of 312,000 could be attained and the resolution was slightly better than that of a light microscope. Bodo von Borries, who had joined Knoll’s group in 1929 as a research student, signs his first paper with Ruska. Borries, B. von and Ruska, E., Die Abbildung durchgestrahlter Folien im Elektronenmikroskop. Z. Physik 83 ~1933! 187–193. Ruska, E., Über Fortschritte im Bau und in der Leistung des magnetischen Elektronenmikroskops. Z. Physik 87 ~1934! 580–602. 1932–1934 Construction of electron microscopes by Ladislaus Marton in Brussels. The first was a “magnifying glass,” with only one lens, but the second instrument, built in 1933, had three lenses and produced the first image of a biological specimen, a 15-mm-thick specimen of the leaf of a sundew plant, impregnated with osmium tetroxide. Marton, L., Electron microscopy of biological objects. Nature 133 ~1934! 911 and Phys. Rev. 46 ~1934! 527– 528. 1934 Publication of the first text in English on electron optics. Martin, L.C., The paraxial equations of electron optics. J. Television Soc. 1 ~1934! 377–383. 1934–1935 E. Driest and H.O. Muller obtain images of unfixed biological specimens ~wings and legs of a housefly! in Ruska’s microscope, now equipped with an internal camera. Soon after, Friedrich Krause imaged diatoms, epithelial cells of the hellbinder, and bacteria. Driest, E. and Muller, H.O., Elektronenmikroskopische Aufnahmen ~Elektronenmikrogramme! von 98 F. Haguenau et al. Chitinobjekten. Z. Wiss. Mikrosk. 52 ~1935! 53–57; Krause, F., Elektronenoptische Aufnahmen von Diatomeen mit dem magnetischen Elektronenmikroskop. Z. Physik 102 ~1936! 417–422; Neuere Untersuchungen mit dem magnetischen Elektronenmikroskop. In Busch, H. and Brüche, E., eds, Beiträge zur Elektronenoptik, pp. 55–61 ~Barth, Leipzig 1937!. 1932 While Knoll and Ruska were developing the transmission electron microscope with magnetic lenses in the TH Berlin, emission microscopes using electrostatic lenses were being developed in the Allgemeine Elektrizitäts-Gesellscahft ~AEG! Research Laboratories, also in Berlin. The first results were obtained by Ernst Brüche and H. Johannson ~1932!. Brüche, E., Elektronenmikroskop. Naturwissenschaften 20 ~1932! 49; Brüche, E. and Johannson, H., Elektronenoptik und Elektronenmikroskop. Naturwissenchaften 20 ~1932! 353–358. Johannson, H., Über das Immersionsobjektiv der geometrischen Elektronenoptik, I, II. Ann. Physik (Leipzig) 18 ~1933! 385–413 and 21 ~1934! 274–284. 1934 Publication of the first monograph on electron optics. Brüche, E. and Scherzer, O., Geometrische Elektronenoptik ~Springer, Berlin 1934!. 1935 Construction of the first North American electron microscope by Paul Anderson and Kenneth Fitzsimmons in Washington State College, Pullman WA. Yoshii, Z., Pioneers of electron microscopy at Washington State University and their work. Bull. Yamaguchi Med. School 17 ~1970! No. 3/4, 191–200; Cohen, A.L. and Steever, R.G.E., An early electron microscope. EMSA Proc. 29 ~1971! 4–5; Reisner, J.H., An early history of the electron microscope in the United States. Adv. Electron. Electron Phys. 73 ~1989! 133–231. 1936 Demonstration by Otto Scherzer that the spherical and chromatic aberration coefficients of electron lenses are intrinsically nonvanishing and hence cannot be eliminated by skillful design. This result was proved by showing that the integrands that occur in the formulae for these coefficients can be written as a sum of squared terms. Scherzer, O., Über einige Fehler von Elektronenlinsen. Z. Physik 101 ~1936! 593–603. 1936 Hans Boersch hints at selected-area diffraction. Boersch, H., Über das primäre und sekundäre Bild im Elektronenmikroskop, I, II. Ann. Physik (Leipzig) 26 ~1936! 631–644 and 27 ~1936! 75–80. 1936 The construction of primitive electron microscopes begins in Japan in 1936, notably by E. Sugata. This work is catalyzed by the creation of a Cooperative Research Committee in 1939 and also by the arrival of a copy of M. von Ardenne’s treatise ElektronenÜbermikroskopie ~Springer, Berlin 1940!. During the war, Hitachi and Shimadzu begin commercial production of electron microscopes; JEOL and Akashi join them in 1949 and 1953, respectively. See The Growth of Electron Microscopy for extensive detail. Kanaya, K., Reminiscences of the development of electron optics and electron microscope instrumentation in Japan. Adv. Electron. Electron Phys., Supplement 16 ~1985! 317–386; Fujita, H., ed., History of Electron Microscopes. Published in commemoration of ICEM-11 ~Kyoto, 1986!; Tadano, B., Progress of electron microscopes in Hitachi. Hitachi Rev. 1 ~1953! No. 4, 19–30. 1937 The Metropolitan-Vickers Company ~Manchester, UK! supplies the first commercial electron microscope ~EM1! to Louis C. Martin at Imperial College, London. The resolution of this instrument, capable of furnishing a light image and an electron image of the same specimen, was no better than that of a light microscope. Martin, L.C., Whelpton, R.V. and Parnum, D.H., A new electron microscope. J. Sci. Instrum. 14 ~1937! 14–24. 1937–1939 E. Ruska and B. von Borries contact Siemens and Carl Zeiss. Siemens & Halske set up an Ultramicroscopy Laboratory in Berlin, and in 1938 two prototype electron microscopes come into operation. A resolution of about 7 nm is achieved. These are used principally for biological studies by Ruska’s brother Helmut and H.O. Müller, who have published some 20 papers by the end of 1939. See the book by Ruska cited below ~1979 and 1980!. 1937 Recognition that brightness has a physical limit. Langmuir, D.B., Theoretical limitations of cathoderay tubes. Proc. Inst. Radio Eng. 25 ~1937! 977–991. History of Electron Microscopy 1938 First use of convergent-beam electron diffraction by Walther Kossel and Gottfried Möllenstedt; this inspires the theoretical work of Carolina MacGillavry. Kossel, W. and Möllenstedt, G., Elektroneninterferenzen in konvergentem Bündel. Naturwiss. 26 ~1938! 660–661; Elektroneninterferenzen im konvergenten Bündel. Ann. Physik 36 ~1939! 113–140; Dynamische Anomalie von Elektroneninterferenzen. Ann. Physik 42 ~1942! 287–293; Möllenstedt, G., Messungen an den Interferenzerscheinungen im konvergenten Elektronenbündel. Ann. Physik 40 ~1941! 39–65. MacGillavry, C., Diffraction of convergent electron beams. Nature 145 ~1940! 189–190; Zur Prüfung der dynamischen Theorie der Elektronenbeugung an Kristallgitter. Physica 7 ~1940! 329–343. 1938 Construction by James Hiller and A. Prebus of a transmission electron microscope with magnetic lenses, once thought to be the first in North America. The useful magnification is 40,0003 and a resolution of 6 nm is achieved. Prebus, A. and Hillier, J., The construction of a magnetic electron microscope of high resolving power. Can. J. Res. 17A ~1939! 49–63. 1938 Construction of the first scanning electron microscope and the first scanning transmission electron microscope by Manfred von Ardenne. 99 Borries, B. von and Ruska, E., Versuche, Rechnungen und Ergebnisse zur Frage des Aulösungsvermögens beim Übermikroskop. Z. Tech. Phys. 20 ~1939! 225– 235. 1939 Hans Mahl and Hans Boersch ~AEG, Berlin! describe electrostatic microscopes. Mahl, H., Über das elektrostatische Elektronenmikroskop hoher Auflösung. Z. Tech. Phys. 20 ~1939! 316–317. Boersch, H., Das Elektronen-Schattenmikroskop. Z. Tech. Phys. 20 ~1939! 346–350. 1939 Manne Siegbahn ~winner of a Nobel prize for his work on X-ray spectroscopy!, builds the first Scandinavian electron microscope, which has a resolution of about 150 nm. In about 1944, Georg Schönander AB in Stockholm produces a commercial model, for which a resolution of 2 nm was claimed, of which 10–15 were constructed. Siegbahn, M., Vetenskapakadamiens Forskningsinstitut för experimentell fysik. Kungl. Svenska Vetenskapsakadamiens Årsbok ~1939! 147–148 @a paragraph of this annual report describes the electron microscope project#; Bergqvist, A., Det Svenska elektronenmikroskopet. Tek. Tidskr. 76 ~1946! 649–655 or Ett Svensk elektronmikroskop. Industritidningen Norden 33 ~1946! 3–7. 1939 Publication of a major treatise on electron optics. von Ardenne, M., Das Elektronen-Rastermikroskop. Theoretische Grundlagen. Z. Physik 109 ~1938! 553– 572, and Das Elektronenmikroskop. Praktische Ausführung. Z. Tech. Phys. 19 ~1938! 407–416. A contemporary description in English is to be found in von Ardenne’s U.S. Patent No. 2,257,774, “ElectronicOptical Device”, filed February 15, 1938 and granted October 7, 1941. 1940 Publication of an influential treatise by von Ardenne. 1939 Siemens delivers the first serially produced transmission electron microscope; between 1939 and 1945, 38 microscopes are constructed. Details of these are listed by C. Wolpers. A paper by von Borries and Ruska shows that a resolution of 7 nm was achieved at 70 kV. Mahl, H., Metallkundliche Untersuchungen mit dem elektrostatischen Übermikroskop. Z. Tech. Phys. 21 ~1940! 17–18; Plastisches Abdruckfahren bei Oberflächen. Z. Tech. Phys. 22 ~1941! 33. Wolpers, C., Electron microscopy in Berlin 1928– 1945. Adv. Electron Electron Phys. 81 ~1991! 211– 229. Myers, L.M., Electron Optics, Theoretical and Practical ~Chapman and Hall, London 1939!. Ardenne, M. von, Elektronen-Übermikroskopie ~Springer, Berlin 1940!. 1940 Introduction of replica techniques into electron microscopy by Hans Mahl. 1940 James Hillier joins RCA ~Camden, NJ! and develops a commercial TEM. Under the lease-lend treaty, six are sent to the United Kingdom. For a few years, RCA dominates the electron microscope market, but after 100 F. Haguenau et al. the war, many European and Japanese firms develop such instruments and in 1969, RCA ceases production of electron microscopes. For a critical account of the RCA contribution, see the book by Rasmussen listed below ~1997!. 1940 Allgemeine Elektrizitäts-Gesellscahft ~AEG! complete a production model of their EM5 electron microscope with electrostatic lenses. Ramsauer, C. ~ed.!, Elektronenmikroskopie. Bericht über Arbeiten des AEG Forschungs-Instituts 1930 bis 1942 ~Springer, Berlin 1943!. 1941 Walter Glaser introduces a bell-shaped model to represent the magnetic field distribution in magnetic lenses. This gives closed-form expressions for all the paraxial optical properties and aberration coefficients of such lenses, a very valuable feature in precomputer days. Glaser, W., Streng Berechnung magnetischer Linsen H0 . Z. Physik 117 ~1941! der Feldform H 5 1 1 ~z/a! 2 285–315. 1941–1944 Construction of electron microscopes by Jan Le Poole in the Technische Hogeschool in Delft. These are the forerunners of the Philips range of electron microscopes. See the contribution by J.B. Le Poole to “The Beginnings of Electron Microscopy” ~1985!, listed below and the memoir of Le Poole by T. Mulvey and D.J.J. van de Laak–Tijssen ~Adv. Imaging & Electron Phys. 115, 2001, 287–354!. 1941–1942 Ruthemann’s work on energy losses foreshadows EELS. Ruthemann, G., Diskrete Energieverluste schneller Elektronen in Festkörpern. Naturwissenschaften 29 ~1941! 648; Elektronenbremsung an Röntgenniveaus. Ibid. 30 ~1942! 145; Diskrete Energieverluste mittelschneller Elektronen beim Durchgang durch dünne Folien. Elektronenbremsung an Röntgenniveaus. Physik 2 ~1948! 113–134 and 135–146. 1942 In France, electron microscopes are constructed by Gaston Dupouy ~Toulouse; magnetic lenses! and by Pierre Grivet ~CSF, Paris; electrostatic lenses!. The MIII CSF instrument is marketed in 1946 and a much improved model, the MIX, is announced in 1949. Optique de Précision de Levallois ~OPL! produce a commercial model of the magnetic microscope in 1955. Dupouy, G., Microscope électronique magnétique à grand pouvoir de résolution. J. Phys. Radium 7 ~1946! 320–329. Grivet, P. and Bruck, H., Le microscope électronique électrostatique. Ann. Radioél. 1 ~1946! 293–310. Fert, C. and Selme, P., Le microscope électronique O.P.L. Bull. Microsc. Appl. 6 ~1956! 157–164. 1942 TEM images of collagen are obtained at MIT ~Cambridge MA!. Schmidt, F.O., Hall, C.E. and Jakus, M., Electron microscope studies of the structure of collagen. J. Cell. Comp. Physiol. 20 ~1942! 11–33. 1942 First example of shadowing by metal evaporation by Heinz Müller, independently introduced in 1944 by Robley Williams and Ralph Wyckoff. Müller, H.O., Die Ausmessung der Tiefe übermikroskopischer Objekte. Kolloid Z. 99 ~1942! 6–28. Williams, R.C. and Wyckoff, R.W.G. ~1944!. The thickness of electron microscopic objects. J. Appl. Phys. 15 ~1944! 712–715. 1943 James Hillier describes the possibility of energy-loss spectroscopy; together with R.F. Baker, he obtains the first inner-shell loss spectrum. Hillier, J., On microanalysis by electrons. Phys. Rev. 64 ~1943! 318–319; Hillier, J. and Baker, R.F., Microanalysis by means of electrons. J. Appl. Phys. 15 ~1944! 663–675. 1944 Development of a Swiss microscope by Giovanni Induni at Trüb-Täuber. The KM model is marketed in 1947. Induni, G., Das Schweizerische Übermikroskop. Vierteljahresschr. Naturforsch. Ges. Zürich 90 ~1945! 181– 195. 1945 Publication of a very influential text. Zworykin, V.K., Morton, C.A., Ramberg, E.G., Hillier, J. and Vance, A.W., Electron Optics and the Electron Microscope ~Wiley, New York 1945!. 1946 Publication of the first full account of Fourier optics, invented by Pierre-Michel Duffieux. History of Electron Microscopy Duffieux, P.-M., L’Intégrale de Fourier et ses Applications à l’Optique ~privately printed for the author by Les Imprimeries Oberthur, Rennes 1946!. 1947–1949 The principle of the stigmator is discovered by François Bertein ~Paris, 1947!, James Hillier ~RCA, 1947! and Otto Rang ~Mosbach, 1949!. Bertein, F., Un système correcteur en optique électronique, C.R. Acad. Sci. Paris 225 ~1947! 801–803; Hillier, J. and Ramberg, E.G., The magnetic electron microscope objective: contour phenomena and the attainment of high resolving power. J. Appl. Phys. 18 ~1947! 48–71; Rang, O., Der elektrostatische Stigmator, ein Korrektiv für astigmatische Elektronenlinsen. Optik 5 ~1949! 518–530. 1947 Invention of the focusing aid known as the “wobbler” by J.B. Le Poole; The Philips EM 100 is equipped with this device. Le Poole, J.B., A new electron microscope with continuously variable magnification. Philips Tech. Rev. 9 ~1947! 33–46. 1947 Otto Scherzer lists almost all the ways of correcting the spherical aberration of electron lenses. Scherzer, O. Sphärische und chromatische Korrektur von Elektronenlinsen, Optik 2 ~1947! 114–132. 1947 The first serially produced English electron microscope appears, the EM2. Several models are produced until 1978, when Associated Electrical Industries ~AEI! ceases electron microscope production. Haine, M.E., The design and construction of a new electron microscope. J. Inst. Elec. Eng. 14 ~1947! 447– 462; The electron optical system of the electron microscope. J. Sci. Instrum. 24 ~1947! 61–66. 101 1948 Invention of holography by Denis Gabor ~Nobel Prize 1971! as a means of circumventing the resolutionlimiting effect of the spherical aberration of electron lenses. Gabor, D., A new microscopic principle. Nature 161 ~1948! 777–778. 1948–1954 Early books in Japanese on electron microscopy and electron optics. Suzuki, S., Denshi Kenbikyo @Electron Microscopy# ~Kawade Shobo, 1948!. Sasagawa, K., Denshi Kenbikyo @Electron Microscopy# ~Honda Shoten, 1951!. Kanaya, K., Denshi Kenbikyo, Riron to sono Toriatsukai @Electron Microscope, Theory and Practice# ~Denki Shoin, Kyoto 1954!. 1948–1953 The development of the ultramicrotome. The arrival of microtomes capable of cutting very thin sections had an immense effect on the development of the subject. It halted the progress towards higher voltage microscopes, which seemed essential for the thick specimens then being examined, and it improved the quality of micrographs out of all recognition. Since the main impact of these new ultramicrotomes was in biology, the references are to be found in Section 4.1. 1949 The first international congress on electron microscopy is held in Delft. The proceedings of this meeting herald many major developments in electron microscopy. Houwink, A.L., Le Poole, J.B. and Le Rütte, W.A. ~eds! Proceedings of the Conference on Electron Microscopy, Delft, 4–8 July, 1949 ~Hoogland, Delft, 1950!. 1948 Charles Oatley launches a programme of research in the Cambridge University Engineering Department that culminates in the appearance of the first commercial scanning electron microscope in 1965. The first such microscope is constructed by Dennis McMullan and the first images are obtained in 1951. The work is continued by Kenneth C.A. Smith, Oliver Wells ~who obtained the first stereo 3-D images! and many others. 1949 First studies on image formation and resolving power in wave-optical terms. McMullan, D., An improved scanning electron microscope for opaque specimens. Proc. Inst. Elec. Eng. 100 ~1953! 245–259. Glaser, W. and Schiske, P., Elektronenoptische Abbildung auf Grund der Wellenmechanik. Ann. Physik 12 ~1953! 240–266 and 267–280. Scherzer, O., The theoretical resolution limit of the electron microscope. J. Appl. Phys. 20 ~1949! 20–29. Glaser, W., Zur wellenmechanischen Theorie der elektronenoptischen Abbildung. Sitzungsber. Öst. Akad. Wiss. Math-Naturw. Kl., Abt IIa, 159 ~1950! 297–360. 102 F. Haguenau et al. Glaser, W. and Braun, G., Zur wellenmechanischen Theorie der elektronenoptischen Abbildung. Acta Phys. Austriaca 9 ~1954–5! 41–74 and 267–296. 1951 Publication by Peter Andrew Sturrock of a formal approach to aberration theory, inspired by Glaser’s pioneering work. 1949 Philips launches its first commercial microscope, the EM100; this is followed by a series of progressively improved models, which continues today ~2002! under FEI management. See Agar in The Growth of Electron Microscopy ~1996! for a full account. Sturrock, P.A., Perturbation characteristic functions and their application to electron optics. Proc. Roy. Soc. (London) A210 ~1951! 269–289. Dorsten, A.C. van, Nieuwdorp, H. and Verhoeff, A., The Philips EM100. Philips. Tech. Repts. 12 ~1950! 33–64. 1949 Publication by Ehrenberg and Siday of an article in which the so-called Aharonov–Bohm effect is predicted on semi-classical grounds. After many years of dispute, the existence of the effect is proved beyond any doubt by Akira Tonomura et al. Ehrenberg, W. and Siday, R.E., The refractive index in electron optics and the principles of dynamics. Proc. Phys. Soc. (London) B62 ~1949! 8–21; Aharonov. Y. and Bohm, D., Significance of electromagnetic potentials in the quantum theory. Phys. Rev. 115 ~1959! 485–491; Tonomura, A., Osakabe, N., Matsuda, T., Kawasaki, T. and Endo, J., Evidence for Aharonov– Bohm effect with magnetic field completely shielded from electron wave. Phys. Rev. Lett. 56 ~1986! 792– 795. 1952 Publication by Walter Glaser of a treatise on electron optics that remained the standard work for many decades. Glaser, W., Grundlagen der Elektronenoptik ~Springer, Vienna 1952!. 1953 A Metropolitan-Vickers EM3, still in its crate, is discovered in Chongquing, China; it is assembled and is the prototype for the first Chinese microscopes. 1953 Appearance of a commercial Czech microscope, the TESLA BS241. Delong, A. and Drahos, V., Ceskoslovensky elektronovy mikroskop. Sb. VST Brno 20 ~1951! 334–348. 1953 A heavily used text by Cecil Hall is published. Hall, C.E., Introduction to Electron Microscopy ~McGraw–Hill, New York 1953!. 1954 Heinz Düker and Gottfried Möllenstedt obtain interference fringes by use of an electron biprism. Möllenstedt, G. and Düker, H., Fresnelsche Interferenzversuche mit einem Biprisma für Elektronenwellen. Naturwissenschaften 42 ~1954! 41–43. 1949–1951 Development of the X-ray microanalyser by Raymond Castaing; preliminary descriptions are to be found in the Proceedings of the Delft ~1949! and Paris ~1950! conferences on electron microscopy and a full account becomes available in 1951. 1954 Discovery of the effect now known as the Boersch effect, whereby the energy distribution in an electron beam is altered if the current density becomes high. Castaing, R., Application des sondes électroniques à une méthode d’analyse ponctuelle chimique et cristallographique. Thèse, Paris 1951; Microanalysis by means of an electron-probe principle and corrections. In Electron Physics, National Bureau of Standards Circular 527 ~1954! 305–308. 1955 A heavily used set of “universal curves” representing magnetic lens properties is published by G. Liebmann. 1951 Resumption of microscope production by Siemens. The ÜM100 appears in 1951 and the Elmiskop 1 ~resolution 1 nm! in 1954; the Elmiskop 101 follows in 1968, the 102 in 1972, and the CT150 in 1976, but in 1979, Siemens discontinues electron microscope production. Boersch, H. Experimentelle Bestimmung der Energieverteilung in thermisch ausgelösten Elektronenstrahlen. Z. Physik 139 ~1954! 115–146. Liebmann, G., A unified representation of magnetic electron lens properties. Proc. Phys. Soc. London B68 ~1955! 737–745. 1955 Publication of a full account in English of the eikonal method of analyzing electron optical systems. Sturrock, P.A., Static and Dynamic Electron Optics ~Cambridge University Press, London 1955!. History of Electron Microscopy 1956 Introduction of the pointed filament by Tadahoshi Hibi. Hibi, T., Pointed filament. I. Its production and its applications. J. Electronmicrosc. 4 ~1956! 10–15. 1956 James Menter observes crystal lattice images with dislocations. Menter, J.W., The direct study by electron microscopy of crystal lattices and their imperfections. Proc. Roy. Soc. (London) A236 ~1956! 119–135. 1956 Scanning coils are added to the microanalyser by Peter Duncumb. @In the version introduced by Castaing, the specimen was moved mechanically under the ~stationary! beam.# In 1960, the Cambridge Instrument Company markets this as the Microscan. Cosslett, V.E. and Duncumb, P.D., Micro-analysis by a flying-spot X-ray method. Nature 177 ~1956! 1172– 1173. 1957 The multislice method of image simulation is proposed by John Cowley and Alexander Moodie and is put into practice by Dennis Lynch and Michael O’Keefe when the necessary computing power becomes available. Cowley, J.M. and Moodie, A.F., Fourier images. I. The point source. Proc. Phys. Soc. London B70 ~1957! 486– 496; The scattering of electrons by atoms and crystals. I. A new theoretical approach. Acta Cryst. 10 ~1957! 609–619; Allpress, J.G., Hewat, E.A., Moodie, A.F. and Sanders, J.V., n-beam lattice images. I. Experimental and computed images from W4Nb26O77 . Acta Cryst. A28 ~1972! 528–536; Lynch, D.F. and O’Keefe, M.A., n-beam lattice images. II. Methods of calculation. Acta Cryst. A28 ~1972! 536–548; Anstis, G.R., Lynch, D.F., Moodie, A.F. and O’Keefe, M.A., n-beam lattice images. III. Upper limits of ionicity in W4Nb26O77 . Acta Cryst. A29 ~1973! 138–147; O’Keefe, M.A. n-beam lattice images. IV. Computed two-dimensional images. Acta Cryst. A29 ~1973! 389–401. 1957 Studies on the effect of inelastic scattering on electron diffraction patterns by H. Yoshioka. Yoshioka, H., Effect of inelastic waves on electron diffraction. J. Phys. Soc. Japan 12 ~1957! 618–628. 1958 The Pulp and Paper Research Institute at Pointe Claire ~Quebec, Canada! takes delivery of a scanning elec- 103 tron microscope, based on the model constructed by K.C.A. Smith in the Department of Engineering of Cambridge University. Ten years later, Rezanowich summarizes its uses. Rezanowich, A., Some applications of the scanning electron microscope at the Pulp and Paper Research Institute of Canada. Scanning Electron Microsc. ~1968! 13–27. 1959–1963 In a series of publications, Peter Hirsch, Archibald Howie, Michael Whelan, and Hatsujiro Hashimoto develop the kinematic and dynamical theory of image formation in crystalline specimens ~for individual references, see Section 3!. This is brought together in a book by Hirsch et al. Hirsch, P.B., Howie, A., Nicholson, R.B., Pashley, D.W. and Whelan, M.J., Electron Microscopy of Thin Crystals ~Butterworths, London 1965!. 1959 Publication of a full treatise on electron optics in Russian. Kel’man, V.M. and Yavor, S.Ya., Elektronnaya Optika ~Izd. Akad. Nauk SSSR, Moscow and Leningrad 1959!. 1959 Major paper by W. Tretner completes Scherzer’s result of 1936 ~that chromatic and spherical aberration can never be eliminated from round lenses by skillful design!; Tretner establishes the limits on these coefficients as a function of physical constraints. Tretner, W., Existenzbereiche rotationssymmetrischer Elektronenlinsen. Optik 16 ~1959! 155–184. 1959 Use of the gas proportional counter for the analysis of light elements in X-ray microanalysis and first explorations of signal processing. Dolby, R.M., Some methods for analysing unresolved proportional counter curves of X-ray line spectra. Proc. Phys. Soc. ~London! 73 ~1959! 81–94; Dolby, R.M. and Cosslett, V.E., A spectrometer system for long-wavelength X-ray emission microanalysis. In X-ray Optics and X-ray Microanalysis ~Engström, A., Cosslett, V. and Pattee, H., eds! 351–357 ~Elsevier, Amsterdam, London & New York, 1960!. 1960 Gaston Dupouy obtains the first pictures with the 1.2-MV microscope, constructed in Toulouse. ~Dupouy had great hopes that this instrument would en- 104 F. Haguenau et al. able biologists to study living material by electron microscopy. For similar attempts at 100 kV, see the work of I.G. Stoyanova, listed with radiation damage, 1970.! Dupouy, G., Perrier, F., and Durrieu, L., L’observation de la matière vivante au moyen d’un microscope électronique fonctionnant sous très haute tension. C.R. Acad. Sci. Paris 251 ~1960! 2836–2841. 1960 Incorporation of argon ion beam etching in a SEM. Stewart, A.D.G., Investigation of the topography of ion bombarded surfaces with a scanning electron microscope. Proc. ICEM-5 ~Philadelphia 1962! vol. 1, D-12. 1960 Description of a much-used SEM detector, the Everhart–Thornley detector. Everhart, T.E. and Thornley, R.F.M., Wideband detector for micro-microampère low-energy electron currents. J. Sci. Instrum. 37 ~1960! 246–248. 1962 Raymond Castaing and Lucien Henry describe an energy filter combining a magnetic prism and an electrostatic mirror for elemental mapping. Castaing, R. and Henry, L., Filtrage magnétique des vitesses en microscopie électronique. C. R. Acad. Sci. Paris 255 ~1962! 76–78. 1964 First proposals for magnetic lenses exploiting superconductivity. The early proposals of André Laberrigue and Paul Levinson in Reims were quickly followed by work in Chicago ~Humberto Fernández-Morán!, Japan ~S. Ozasa et al.!, Berlin ~Hans Boersch!, and in the Siemens Research Laboratory in Munich. A section of the proceedings of the International Congress on Electron Microscopy held in Kyoto in 1966 is devoted to superconducting lenses. A major development was the introduction of the “shielding lens” by Isolde Dietrich and colleagues in Munich. The subject was reviewed by David Hardy in 1973 and the book by Isolde Dietrich may be regarded as its swan song. electron microscopy with superconducting lenses at liquid helium temperatures. Ibid. 56 ~1966! 801– 808. Boersch, H., Bostanjoglo, O. and Grohmann, K., Supraleitender Hohlzylinder als magnetische Linse. Z. Angew. Physik 20 ~1966! 193–194. Ozasa, S., Katagiri, S., Kimura, H. and Tadano, B., Superconducting electron lens. ICEM-6 ~Kyoto 1966! 1, 149–150. Dietrich, I., Weyl, R. and Zerbst, H., High magnetic field gradient for electron microscopy. Cryogenics 7 ~1967! 178–179. Dietrich, I., Pfisterer, H. and Weyl, R., Eine supraleitende Linse für das Elektronenmikroskop. Z. Angew. Phys. 28 ~1969! 35–39. Weyl, R., Dietrich, I. and Zerbst, H., The superconducting shielding lens. Optik 35 ~1972! 280–286. Hardy, D.F., Superconducting electron lenses. Adv. Opt. & Electron Microsc. 5 ~1973! 201–237. Dietrich, I., Superconducting Electron-optic Devices. ~Plenum, New York & London 1976!. 1965 The Cambridge Instrument Company launches the first commercial SEM, the Stereoscan. The first full account of the modern SEM is prepared by Charles Oatley, William Nixon, and Fabian Pease. Stewart, A.D.G. and Snelling, M.A., A new scanning electron microscope. Proc. EUREM-3 ~Prague 1964! A, 55–56. Oatley, C.W., Nixon, W.C. and Pease, R.F.W., Scanning Electron Microscopy. Adv. Electron. Electron Phys. 21 ~1965! 181–247. Laberrigue, A. and Levinson, P., Utilisation des propriétés des fils supraconducteurs en microscopie électronique. C. R. Acad. Sci. Paris 259 ~1964! 530–532. 1965–1966 At a conference in Cambridge in 1965 and at ICEM-6 ~Kyoto, 1966!, Albert Crewe describes briefly the scanning transmission electron microscope ~STEM!; a successful working model is presented in 1968. This is the first major use of a field-emission gun in electron microscopy. The gun was designed by James Butler, an early example of computer-aided design in electron optics. Fernández-Morán, H., Electron microscopy with high-field superconducting solenoid lenses. Proc. Natl. Acad. Sci. USA 53 ~1965! 445–451; High-resolution Crewe, A.V., Wall, J. and Welter, L.M., A high-resolution scanning transmission electron microscope. J. Appl. Phys. 39 ~1968! 5861–5868. History of Electron Microscopy 105 Butler, J.W., Digital computer techniques in electron microscopy. ICEM-6 ~Kyoto! 1, 191–192. placed on a firm theoretical footing by Anthony Crowther soon after. 1965 Karl-Joseph Hanszen introduces the notion of contrasttransfer function into electron optics. Rosier, D. de and Klug, A., Reconstruction of three dimensional structures from electron micrographs. Nature 217 ~1968! 130–134. Hanszen, K.-J. and Morgenstern, B., Die Phasenkontrast- und Amplitudenkontrast-Übertragung des elektronenmikroskopischen Objektivs. Z. Angew. Phys. 19 ~1965! 215–227; Hanszen, K.-J., Problems of image interpretation in electron microscopy with linear and nonlinear transfer. Z. Angew. Phys. 27 ~1969! 125–131. 1965–1966 Completion of the Cambridge high-voltage electron microscope ~750 kV! and several Japanese 1-MV microscope projects. See the proceedings of ICEM-6 ~Kyoto, 1966!. 1966 First practical use of the condenser–objective lens, the advantages of which had been predicted by Glaser. Riecke, W.D. and Ruska, E., A 100 kV transmission electron microscope with single-field condenser– objective. ICEM-6 ~Kyoto 1966! 1, 19–20. 1966 Visualization of the contrast-transfer function by Thon. Thon, F., Zur Defokussierunsabhängigkeit des Phasenkontrastes bei der elektronenmikroskopischen Abbildung. Z. Naturforsch. 21a ~1966! 476–478. 1966 Arrival of the Metals Research “Quantimet” for the analysis of scanning electron microscope images. This really comes of age with the Model 720, in 1972, which provides high-speed, dedicated, automatic digital image analysis and external digital scan control of the SEM. Cole, M., The Metals Research Quantimet ~QTM!. Microscope & Crystal Front 15 ~1966! 148–160; Fisher, C., The new Quantimet 720. Microscope 19 ~1972! 1–20; Fisher, C., Automatic image analysis for the stereologist. J. Microscopy 95 ~1972! 385–392. 1968 First optical reconstruction of an in-line electron hologram by Akira Tonomura et al. Tonomura, A., Fukuhara, A., Watanabe, H. and Komoda, T., Optical reconstruction of image from Fraunhofer electron-hologram. Japan. J. Appl. Phys. 7 ~1968! 295. 1968 First three-dimensional reconstruction ~of a virus! by David de Rosier and Aaron Klug. The method was Crowther, R.A., Procedures for three-dimensional reconstruction of spherical viruses by Fourier synthesis from electron micrographs. Phil. Trans. Roy. Soc. London B261 ~1971! 221–230. 1968–1970 First experiments on off-axis holography by Gottfried Möllenstedt and Herbert Wahl in Tübingen and Akira Tonomura in the Hitachi Advanced Research Laboratory ~Tokyo!. Möllenstedt, G. and Wahl, H., Elektronenholographie und rekonstruktion mit Laserlicht. Naturwissenschaften 55 ~1968! 340–341; Tonomura, A., Electron beam holography. J. Electron Microsc. 18 ~1969! 77–78. 1968 First attempts to process scanning microscope images. MacDonald, N.C., Computer-controlled scanning electron microscopy. EMSA Proc. 26 ~1968! 362–363; White, E.W., McKinstry, H.A. and Johnson, G.G., Computer processing of SEM images. Scanning Electron Microsc. ~1968! 95–103. 1968 Description of the first analytical TEM designed for X-ray spectroscopy. Ahead of its time, but constrained by the use of bulky, inefficient crystal spectrometers, the EMMA was not a commercial success, but paved the way for the next generation of EDS-based AEMs. Duncumb, P., EMMA, A combination of an electron microscope and a microprobe analyser, J. Microscopie 7, 581–589. 1968 Reliable scattering factor tables are published. Doyle P.A. and Turner P.S., Relativistic Hartree–Fock X-ray and electron scattering factors. Acta Cryst. A24, 390–397. 1968 First book on scanning electron microscopy. Thornton, P.R., Scanning Electron Microscopy, Applications to Materials and Device Science ~Chapman & Hall, London 1968!. 1969 Notion of ptychography introduced by Walter Hoppe. In this technique, information about the phase distri- 106 F. Haguenau et al. bution of the electron wavefunction is obtained by modulating the wave incident on the specimen. Hoppe, W., Beugung in inhomogenen Primärstrahlwellenfeld. I. Acta Cryst. A25 ~1969! 495–501; Hoppe, W. and Strube, G., II. Acta Cryst. A25, 502–507; Hoppe, W., III. Acta Cryst. A25 ~1969! 508–514. 1969 Addition of a post-column electron energy-loss spectrometer. Wittry, D.B., An electron spectrometer for use with the transmission electron microcope. J. Phys. D2 ~1969! 1757–1766. 1970 First images obtained with the Toulouse 3-MV microscope presented at the ICEM in Grenoble. Dupouy, G. and Perrier, F., Microscope électronique 3 millions de volts. ICEM-7 ~Grenoble, 1970! 1, 129– 130. 1970 First full study of the mechanism of STEM image formation. Thomson, M.G.R. and Zeitler, E., Scanning transmission electron microscopy. I and II. Optik 31 ~1970! 258–280 and 359–366. 1970 Radiation damage ~that is, damage to the specimen caused by electron bombardment! becomes a major preoccupation. The following selection gives an idea of the attempts to study and circumvent it, including some early work on the study of living material in a microchamber ~see also Dupouy, 1960!. Stojanova, I.G. and Belawzewa, E.M., Experimentelle Untersuchung der thermischen Einwirkung des Elektronenstrahls auf das Objekt im Elektronenmikroskop. ICEM-4 ~Berlin, 1958! vol. 1, 100–103. Stoyanova, I.G., Nekrasova, T.A. and Biryuzova, V.I., Study of the action of radiation on bacterial cells in the wet microchamber of an electron microscope. Dokl. Akad. Nauk 131 ~1960! 195–198; Sov. Phys. Dokl. 5 ~1960–1! 433–436. Stoyanova, I.G. and Nekrasova, T.A., Study of live microorganisms in the electron microscope by the gas microchamber method. Dokl. Akad. Nauk 134 ~1960! 467–470; Sov. Phys. Dokl. 5 ~1960–1961! 1117–1121. Stoyanova, I., On damage of electron microscope objects by electrons of average energies; Some results obtained while studying bacterial cells in gas microchamber of electron microscope. ICEM-6 ~Kyoto, 1966! 1, 581–582 and 2, 265–266. Stenn, K. and Bahr, G.F., Specimen damage caused by the beam of the transmission electron microscope, a correlative reconsideration. J. Ultrastruct. Res. 31 ~1970! 526–550. Grubb, D.T. and Groves, G.W., Rate of damage of polymer crystals in the electron microscope. Dependence on temperature and beam voltage. Phil. Mag. 24 ~1971! 815–828. Baumeister, W., Hahn, M., Seredynski, J. and Herbertz, L.M., Radiation damage of proteins in the solid state: changes of amino acid composition in catalase. Ultramicroscopy 1 ~1976! 377–382. Reimer, L., Information about the radiation damage of organic molecules by electron diffraction. J. Microsc. Spectrosc. Electron. 3 ~1978! 579–590. Glaeser, R.M. and Taylor, K.A., Radiation damage relative to transmission electron microscopy of biological specimens at low temperature: A review. J. Microscopy 112 ~1978! 127–138. “Cryomicroscopy and radiation damage.” Special issue of Ultramicroscopy 10 ~1982! 1–177. 1970 Revival of interest in convergent-beam electron diffraction. Beauvillain, J., Formation simultanée de deux diagrammes de diffraction électronique. J. Microscopie 9 ~1970! 455–464. 1970 The first description of the use of twin ion guns to thin nonconducting specimens from both sides simultaneously. Also the first paper to describe rotating the specimen to give more uniform surface finish. From this paper has grown the current broad spectrum of commercial ion-beam thinning devices, essential for TEM of multiphase, insulating, and semiconducting materials. Barber, D.J., Thin films of non-metals made for electron microscopy by sputter etching, J. Mater. Sci. 5, ~1970! 1–8. 1970 By forming the ratio of the elastic and inelastic images in a STEM, Crewe et al. present images of History of Electron Microscopy single atoms. The contrast mechanism is known as “Z-contrast,” since the ratio of the scattering cross sections is approximately proportional to the atomic number Z. Crewe, A.V., Wall, J. and Langmore, J., Visibility of single atoms. Science 168 ~1970! 1338–1340. 1971 Introduction of the “moving objective lens” by Hajime Ohiwa. Ohiwa, H., Goto, E. and Ono, A., Elimination of third-order aberrations from electron-beam scanning systems. Electron. Commun. Japan 54B ~1971! No. 12, 44–51. 1972 Real-time, direct-view stereoscopic scanning electron microscopy enables microdissection and 3-D video recording to be performed in the scanning electron microscope. Boyde, A., Quantitative photogrammetric analysis and qualitative stereoscopic analysis of SEM images. J. Microscopy 98 ~1973! 452–471; Boyde, A., Real-time stereo TV-speed scanning electron microscopy. Beiträge zur Elektronenmikroskopische Direktabbildung von Oberflächen (BEDO) 7 ~1974! 221–230. 1972 Description of an algorithm for the iterative solution of the phase problem by Ralph Gerchberg and Owen Saxton. Gerchberg, R.W. and Saxton, W.O., A practical algorithm for the determination of phase from image and diffraction plane pictures. Optik 35 ~1972! 237–246. 1972 The STEM begins to be used for molecular weight determination. The first proposal by Joseph Wall is taken up elsewhere, notably by Andreas Engel. Wall, J., Mass and mass loss measurements on DNA and fd phage. EMSA 30 ~1972! 186–187. Engel, A., Molecular weight determination by scanning transmission electron microscopy. Ultramicroscopy 3 ~1978! 273–281. 1973 First full account by Eric Munro of the advantages of the finite-element method for electron optical field calculation. Munro, E., Computer-aided design of electron lenses by the finite-element method. In Image Processing and Computer-aided Design in Electron Optics ~P.W. Hawkes, 107 ed.! pp. 284–323 ~Academic Press, London & New York 1973!. 1974 Publication of the first of a series of books on scanning electron microscopy by Ludwig Reimer. Reimer, L. and Pfefferkorn, G., Raster-Elektronenmikroskopie ~Springer, Berlin & New York 1973, 1977!. 1974 The V-filter is introduced by Harald Rose and Erich Plies. Further studies are made by Gerard Zanchi, Jean-Philippe Pérez, Yolande Kihn, and Jean Sevely, which lead to the construction of such a filter for the 1.2-MV microscope in Toulouse, and by Pearce-Percy et al. Rose, H. and Plies, E., Entwurf eines fehlerarmen magnetischen Energie-Analysators. Optik 40 ~1974! 336–341; Zanchi, G., Perez, J.-P. and Sevely, J., Adaptation of a magnetic filtering device on a one megavolt electron microscope. Optik 43 ~1975! 495–501; Pearce-Percy, H.T., Krahl, D. and Jaeger, J., A 4-magnet imaging spectrometer for a fixed-beam transmission electron microscope. EUREM-6 ~Jerusalem, 1976! 1, 348–349; Zanchi, G., Kihn, Y. and Sevely, J., On aberration effects in the chromatic plane of the V filter. Optik, 60 ~1982! 427–436. 1974 N.H. Dekkers and H. de Lang show that the use of detectors that are not circularly symmetric in STEM yields phase information. Dekkers, N.H. and Lang, H. de, Differential phase contrast in a STEM. Optik 41 ~1974! 452–456. 1974 Vacuum Generators makes a commercial STEM, the HB5. Soon after, Siemens also markets a STEM, the ST100. Wardell, I.R.M., Morphew, J. and Bovey, P., Results and performance of a high-resolution STEM. In Scanning Electron Microscopy; Systems and Applications 1973 ~Nixon, W.C., ed.! 182–185 ~Institute of Physics, London 1973!. Krisch, B., Müller, K.H., Schliepe, R., Thon, F. and Willasch, D., Elmiskop ST100—ein DurchstrahlungsRasterelektronenmikroskop höchster Leistung. Siemens Z. 50 ~1976! 47–50. 1974 Marked revival of interest in low-voltage scanning electron microscopy. 108 F. Haguenau et al. Pawley, J.B., Low voltage scanning electron microscopy. J. Microscopy 136 ~1984! 45–68. introducing a plane of antisymmetry in each half of the filter. Welter, L.M. and Coates, V.J., High resolution scanning microscopy at low accelerating voltages. Scanning Electron Microsc. ~1974! 59–66. Rose, H., Aberration correction of homogeneous magnetic deflection systems. Optik 51 ~1978! 15–38. “Low voltage SEM,” special issue of J. Microscopy 140 ~1985! No. 3. Reimer, L., Image Formation in Low-voltage Scanning Electron Microscopy ~SPIE Press, Bellingham, WA 1993!. 1976 The extraction of crystallographic information from convergent-beam diffraction patterns. Buxton, B.F., Eades, J.A., Steeds, J.W. and Rackham, G.M., The symmetry of electron diffraction zone-axis patterns. Phil. Trans. Roy. Soc. London A281 ~1976! 181–184. 1976 First book wholly on electrostatic lenses. Harting, E. and Read, F.H., Electrostatic Lenses ~Elsevier, Amsterdam 1976!. 1977 Development of parallel EELS ~PEELS!. Jones, B.L., Jenkins, D.G., Booker, G.R. and Fry, P.W., Use of silicon linear photodiode arrays for detection of high-energy electrons. In Developments in Electron Microscopy and Analysis, 1977 ~Misell, D.L., ed.! 73–76 ~Institute of Physics, Bristol 1977!; Johnson, D.E., Csillag, S., Monson, K.L. and Stern, E.A., A photodiode, parallel detection system for energy loss spectroscopy. Proc. EMSA 39 ~1981! 370–371; Shuman, H., Parallel recording of electron energy loss spectra. Ultramicroscopy 6 ~1981! 163–167; Egerton, R.F., Parallel-recording systems for electron energy loss spectroscopy ~EELS!. J. Electron Microsc. Techn. 1 ~1984! 37–52; Egerton, R.F. and Crozier, P.A., A compact parallel-recording detector for EELS. J. Microscopy 148 ~1987! 157–166; Krivanek, O.L., Ahn, C.C. and Keeney, R.B., Parallel detection electron spectrometer using quadrupole lenses. Ultramicroscopy 22 ~1987! 103–115; Krivanek, O.L. and Kundmann, M.K., Progress in parallel-detection EELS. EMAG–MICRO 89 ~London, 1989! 1, 33–40. 1978 Cancellation of some aberrations in V-type filters by the introduction of additional symmetries. The advantages of a symmetric design are now supplemented by 1979 Publication by Ernst Ruska of a history of the early years of the electron microscope. Ruska, E. “Die frühe Entwicklung der Elektronenlinsen und der Elektronenmikroskopie”, Acta Hist. Leopoldina No. 12 ~1979!; English translation by T. Mulvey, The Early Development of Electron Lenses and Electron Microscopy ~Hirzel, Stuttgart 1980! and Microscopica Acta Supplement 5 ~1980!. 1979 The spherical aberration of an objective lens is corrected by holography, as in Gabor’s original proposal. Tonomura, A., Matsuda, T., and Endo, J., Sphericalaberration correction of an electron lens by holography. Japan. J. Appl. Phys. 18 ~1979! 1373–1377. 1979 Explanation of the mode of image formation in the scanning electron microscope with a modest vacuum. Danilatos, G.D. and Robinson, V.N.E., Principles of scanning electron microscopy with high specimen chamber pressures. Scanning 2 ~1979! 72–82. 1979 First intimations of a new form of Z-contrast in the STEM, later to evolve into the high-angle angular dark-field ~HAADF! technique, especially in the hands of Steven Pennycook and Peter Nellist. A seminal article by A. Howie is listed in Section 3. Treacy, M.M.J., Howie, A. and Pennycook, S.J., Z contrast of supported catalyst particles in the STEM. In Electron Microscopy and Analysis 1979 ~Mulvey, T., ed.!, 261–264 ~Institute of Physics, Bristol & London 1980!. Pennycook, S.J., Z-contrast STEM for materials science. Ultramicroscopy 30 ~1989! 58–69. Pennycook, S.J. and Jesson, D.E., High-resolution incoherent imaging of crystals. Phys. Rev. Lett. 64 ~1990! 938–941. Nellist, P.D. and Pennycook, S.J., The principles and interpretation of annular dark-field Z-contrast imaging. Adv. Imaging & Electron Phys. 113 ~2000! 147– 203. History of Electron Microscopy 109 1980 Introduction of the Philips TWIN lens for the EM400 series of transmission electron microscopes. analysis techniques. J. Mol. Biol. 220 ~1991! 877– 887. Mast, K. van der, Rakels, C.J. and Le Poole, J.B., A high quality multipurpose objective lens. EUREM-7 ~The Hague 1980! 1, 72–73; Bormans, B. and Hagemann, P., The TWIN lens system—a high resolution objective lens at 300 kV. Proc. EMSA 44 ~1986! 610–613. 1982 First full description of a large family of unusual magnetic lens designs. 1980 Continuation of work on the extraction of crystallographic information from convergent-beam electron diffraction patterns with a large angular view. 1982 Introduction of Lowicryl resins for low-temperature embedding. Tanaka, M., Saito, R., Ueno, K. and Harada, Y., Largeangle convergent-beam electron diffraction. J. Electron Microsc. 29 ~1980! 408–412 ~see also the series of books listed below, 1983!. 1980 Correspondence analysis is introduced by Joachim Frank and Marin van Heel for image classification. Frank, J. and Heel, M. van, Intelligent averaging of single molecules using computer alignment and correspondence analysis. I. The basic method. EUREM-7 ~The Hague, 1980! 2 ~1980! 690–691; Heel, M. van and Frank, J., Intelligent averaging of single molecules using computer alignment and correspondence analysis. II. Localization of image features. EUREM-7 ~The Hague, 1980! 2 ~1980! 692–693; Heel, M. van and Frank, J., Use of multivariate statistics in analysing the images of biological macromolecules. Ultramicroscopy 6 ~1981! 187–194; Frank, J., Verschoor, A. and Boublik, M., Multivariate statistical analysis of ribosome electron micrographs. L and R lateral views of the 40S subunit from HeLa cells. With an Appendix by Frank, J. and Heel, M. van, Correspondence analysis of aligned images of biological particles. J. Mol. Biol. 161 ~1982! 107–137; Radermacher, M., Wagenknecht, T., Verschoor, A. and Frank, J., A new 3-D reconstruction scheme applied to the 50S ribosomal subunit of E. coli. J. Microscopy 141 ~1986! RP1–RP2; Radermacher, M., Wagenknecht, T., Verschoor, A. and Frank, J., Three-dimensional reconstruction from a singleexposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli. J. Microscopy 146 ~1987! 113–136; Radermacher, M., Threedimensional reconstruction of single particles from random and nonrandom tilt series. J. Electron Microsc. Tech. 9 ~1988! 359–394; Heel, M. van, A new family of powerful multivariate statistical sequence Mulvey, T., Unconventional lens design. In Magnetic Electron Lenses ~P.W. Hawkes, ed.!, pp. 359–420 ~Springer, Berlin 1982!. Carlemalm, E., Garavito, R.M. and Villiger, W., Resin development for electron microscopy and an analysis of embedding at low temperature. J. Microscopy 126 ~1982! 123–143. 1982 First edition of an influential text on high-resolution electron microscopy. Spence, J.C.H., Experimental High-resolution Electron Microscopy ~Oxford University Press, New York and Oxford 1980, 1988, 2002!. 1983 First publication of an atlas of EELS spectra. Ahn, C.C. and Krivanek, O.L., EELS Atlas ~Gatan, Warrendale, PA 1983; Wiley, New York & Chichester 2003!. 1984 The two major contributors to the renaissance of convergent-beam diffraction produce sets of reference images and related data; in the case of the Japanese group, this is the first of a series. Convergent Beam Electron Diffraction of Alloy Phases, assembled by the Bristol Group ~Adam Hilger, Bristol & Boston 1984!. Tanaka, M. and Terauchi, M., Convergent-beam Electron Diffraction ~JEOL, Tokyo 1985!; further volumes appeared ~with Kaneyama, T.! in 1988 and 1994 and ~with K. Tsuda and K. Saitoh! in 2002. 1985 Publication of a collection of historical articles by the pioneers of electron optics and microscopy. P.W. Hawkes ~ed.!, “The Beginnings of Electron Microscopy,” Adv. Electron Electron Phys. Supplement 16 ~1985!. 1985 First English text by Ludwig Reimer on SEM. Reimer, L., Scanning Electron Microscopy ~Springer, Berlin & New York 1985, 1998!. 110 F. Haguenau et al. 1986 Practical high-quality designs of V-filters. 1993 First book on electron holography by Akira Tonomura. Lanio, S., High-resolution imaging magnetic energy filters with simple structure. Optik 73 ~1986! 99–107. Tonomura, A., Electron Holography ~Springer, Berlin & New York 1993!. Lanio, S., Rose, H. and Krahl, D., Test and improved design of a corrected imaging magnetic energy filter. Optik 73 ~1986! 56–68. 1994 Remarkable example of electron crystallography of two-dimensional crystals. 1986 Resumption of holography in Tübingen. Lichte, H., Electron holography approaching atomic resolution. Ultramicroscopy 20 ~1986! 293–304. 1986 Appearance of the standard reference on EELS by Ray Egerton. Egerton, R.F., Electron Energy-loss Spectroscopy in the Electron Microscope ~Plenum, New York and London 1986, 1996!. 1989 Computer-controlled microscopy has been gradually developing for several years. A typical contribution is an article by Koster et al. Koster, A.J., Ruijter, W.J. de, Bos, A. van den and Mast, K. van der, Autotuning of a TEM using minimum electron dose. Ultramicroscopy 27 ~1989! 251–272. 1989 The advantages of combining magnetic and electrostatic lenses are explained. Frosien, J., Plies, E. and Anger, K., Compound magnetic and electrostatic lenses for low-voltage applications. J. Vac. Sci. Technol. B7 ~1989! 1874–1877. 1989 Introduction by John Rodenburg of a technique for solving the phase problem by recording all the information furnished by a STEM. Rodenburg, J.M., Higher spatial resolution via signal processing in the microdiffraction plane. EMAG– MICRO 89 ~London 1989! 1, 103–106; The phase problem, microdiffraction and wavelength-limited resolution—a discussion. Ultramicrocopy 27 ~1989! 413–422. 1993 Description of the design concepts that led to a very successful series of transmission electron microscopes, exploiting the benefits of a high-coherence, highbrightness electron source. Otten, M.T. and Coene, W.M.J., High-resolution imaging on a field-emission TEM, Ultramicroscopy 48, 77–91. Kühlbrandt, W., Wang, D.N. and Fujiyoshi, Y., Atomic model of plant light-harvesting complex by electron crystallography. Nature 367 ~1994! 614–621. 1994 The beginning of telemicroscopy. Ellisman, M.H., Soto, G.E. and Martone, M.E., The merger of microscopy and advanced computing: a new frontier for the 21st century. Proc. MSA 52 ~1994! 10–11; Zaluzec, N.J., Tele-presence microscopy/ LabSpace: An interactive collaboratory for use in education and research. Proc. MSA 54 ~1996! 382–383. 1996 Publication of a collection of historical articles by representatives of many of the member societies of IFSEM ~International Federation of Societies of Electron Microscopy!. T. Mulvey ~ed.!, “The Growth of Electron Microscopy”. Adv. Imaging Electron Phys. 96 ~1996!. 1996 Publication of an authoritative teaching text on electron microscopy. Williams, D.B. & Carter, C.B., Transmission Electron Microscopy, A Textbook for Materials Science ~Plenum, New York and London 1996!. 1997 Two attempts to correct spherical aberration are successful: Ondrej Krivanek uses quadrupoles and octopoles to reduce the size of a STEM probe in a commercial instrument and Max Haider uses a sextupole combination to correct the aberrations of the objective lens of a TEM. Krivanek, O.L., Dellby, N., Spence, A.J., Camps, R.A. and Brown, L.M., Aberration correction in the STEM. EMAG 97, 35–40; Krivanek, O.L., Dellby, N., and Lupini, A.R., Towards sub-Å electron beams. Ultramicroscopy 78 ~1999! 1–11; Batson, P.E., Dellby, N. and Krivanek, O.L., Sub-ångström resolution using aberration corrected electron optics. Nature 418 ~2002! 617–620. Haider, M. and Uhlemann, S., Seeing is not believing: reduction of artefacts by an improved point resolu- History of Electron Microscopy tion with a spherical aberration corrected 200kV transmission electron microscope. Microsc. & Microanal. 3 ~1997! Suppl. 2, 1179–1180; Haider, M., Uhlemann, S., Schwan, E., Rose, H., Kabius, B. and Urban, K., Electron microscopy image enhanced. Nature 392 ~1998! 768–769; Haider, M., Rose, H., Uhlemann, S., Schwan, E., Kabius, B. and Urban, K., A sphericalaberration-corrected 200 kV transmission electron microscope. Ultramicroscopy 75 ~1998! 53–60. 2000 Description of the ambitious SuperSTEM project, in which two corrected STEMs will be constructed. Brown, L.M. and Bleloch, A.L., New projects for SuperSTEM. Microsc. Microanal. 6 ~2000! Suppl. 2, 98–99. 2002 Full description of the SMART ~Spectro-Microscope for All Relevant Techniques! project. Hartel, P., Preikszas, D., Spehr, R., Müller, H. and Rose, H., Mirror corrector for low-voltage electron microscopes. Adv Imaging & Electron Phys. 120 ~2002! 41–133. 1997 Direct method of inverting electron diffraction patterns to obtain structure factors proposed by John Spence. Spence, J.C.H., Direct inversion of dynamical electron diffraction patterns to structure factors. Acta Cryst. A54 ~1998! 7–18. 1997 The SEM becomes intelligent. Caldwell, N.H.M, Breton, B.C. and Holburn, D.M., Towards the intelligent SEM. EMAG ~Cambridge 1997! 53–56. 1997 Professional historians of science become interested in electron microscopy. Rasmussen, N., Picture Control. The Electron Microscope and the Transformation of Biology in America, 1940–1960 ~Stanford University Press, Stanford 1997!. 1998 With the defeat of spherical aberration, attention focuses on the energy spread of the electron beam. A number of monochromators are explored. Kahl, F. and Rose, H., Outline of an electron monochromator with small Boersch effect. ICEM-14 ~Cancún 1998! 1, 71–72; Design of a monochromator for electron sources. EUREM-12 ~Brno 2000! 3, I459– I460. Mook, H.W. and Kruit, P., On the monochromatisation of high brightness sources for electron microscopy. Ultramicroscopy 78 ~1999! 43–51; Construction and characterisation of the fringe-field monochromator for a field-emission gun. Ultramicroscopy 81 ~2000! 129–139. Tiemeijer, P.C., Measurement of Coulomb interactions in an electron beam monochromator. Ultramicroscopy 78 ~1999! 53–62; Operation modes of a TEM monochromator. Proc. EMAG ~1999! 191–194. 111 3. E LECTR ON M ICR OSCOPY M ATERIALS S CIENCE IN P HYSICS AND The primary contribution of the transmission electron microscope to materials science and engineering has been in the imaging and analysis of crystalline defects. Such defects, particularly those associated with dislocations, precipitates and their interfaces, and grain boundaries, govern most of the properties ~mechanical, physical, and chemical! of metals, alloys, ceramics, composites, and, in some cases, semiconductors. Accordingly, rather than attempting to list the critical papers in all the different types of materials in the physical sciences, we have emphasized the study of defects as the archetypal application of TEM to the solution of materials problems. We also list several key articles on TEM imaging techniques, electron scattering, TEM diffraction, and analytical techniques. 1928 The first explanation of the occurrence of what we now call Kikuchi lines, which remain the most useful aspect of electron diffraction patterns. Kikuchi, S., Diffraction of cathode rays by mica, and Further study on diffraction of cathode rays by mica. Proc. Imp. Acad. (Tokyo) 4 ~1928! 271–274 and 275– 278. 1949 A truly pioneering paper describes the first use of TEM to study defects in crystals. It uses Bethe’s solution of the Schrödinger equation and provides an explanation of the image contrast of thickness fringes, bend contours, grains, and grain boundaries. Heidenreich, R.D., Electron microscope and diffraction study of metal crystal textures by means of thin sections. J. Appl. Phys. 20 ~1949! 993–1010. 112 F. Haguenau et al. 1957 Development of the “Darwin–Howie–Whelan” equations with the aid of which crystal images could be interpreted. Thomas, G. and Whelan, M.J., Observations of precipitation in thin foils of aluminium 1 4% copper alloy, Phil. Mag. 6 ~1961! 1103–1114. Whelan, M.J. and Hirsch, P.B., Electron diffraction from crystals containing stacking faults. Phil. Mag. 2 ~1957! 1121–1142 and 1303–1324. 1962 Extension of the dynamical theory of electron diffraction to include any small distortion within crystals. Hirsch, P.B., Howie, A. and Whelan, M.J., A kinematical theory of diffraction contrast of electron transmission images of dislocations and other defects. Phil. Trans. Roy. Soc. A252 ~1960! 499–529. Howie, A. and Whelan, M.J., Diffraction contrast of electron microscope images of crystal lattice defects. II. The development of a dynamical theory. III. Results and experimental confirmation of the dynamical theory of dislocation image contrast. Proc. Roy. Soc. London A263 ~1961! 217–237 and A267 ~1962! 206– 230. Hashimoto, H., Howie, A., and Whelan, M.J., Anomalous electron absorption effects in metal foils: Theory and comparison with experiment. Proc. Roy. Soc. London A269 ~1962! 80–103. 1958 One of the first attempts to explain the intensity of electron diffraction patterns through the concept of the dispersion surface @for the determined reader only!#. Kato, N., The flow of X-rays and material waves in ideally perfect single crystals. Acta. Cryst. 11 ~1958! 885–887. 1960 Polymers, a highly innovative use of TEM. At the time, polymer single crystals were first grown from solution; here, for the first time, nonpolyethylene single crystals were grown from dilute solution and both the crystal morphology and also the unit cell structure were elucidated, using TEM and selectedarea electron diffraction. This paper has had a wide impact on many other polymer scientists. Geil, P.H., Nylon single crystals. J. Polymer Sci. 44 ~1960! 449–458. 1961 Complete description of the evidence for the sequence of metastable precipitates that account for the phenomenon of precipitation hardening—the method by which most engineering materials gain their mechanical strength. Takagi, S., Dynamical theory of diffraction applicable to crystals with any kind of small defect. Acta Cryst. 15 ~1962! 1311–1312. 1963 The first description of the detailed rules for interpreting the fringe images of tilted planar defects under different diffracting conditions. Gevers, R., Art, A. and Amelinckx, S., Electron microscope images of single and intersecting stacking faults in thick foils. Phys. Stat. Sol. 3 ~1963! 1563–1593. 1963 Development of a method of obtaining the sign and magnitude of strain parameters in spherical inclusions, prismatic loops and precipitates; widely used since by the TEM community. Ashby, M.F. and Brown, L.M., Diffraction contrast from spherically symmetrical coherency strains. Phil. Mag. 8 ~1963! 1083–1103; On diffraction contrast from inclusions. Phil. Mag. 8 ~1963!1649–1676. 1964 In the mid-1960s, a number of papers show how “resolution” in lattice images could be significantly improved by tilting the incident beam so as to place the primary and one or more diffracted beams equidistant from the optic axis. The technique is exploited by a several groups ~see, e.g., Philips 1973, below! in the study of dislocations and other defects. Komoda, T., On the resolution of the lattice imaging in the electron microscope. Optik 21 ~1964! 93–110. 1964 The practical value of Kikuchi lines is clearly demonstrated, and their presence transforms inaccurate SAD patterns into useful crystallographic orientation tools. Heimendahl, M. von, Bell, W. and Thomas, G., Applications of Kikuchi line analyses in electron microscopy. J. Appl. Phys. 35 ~1964! 3614–3616. 1965 Formal description of the cause of the extinction lines and crosses in CBED patterns, from which it is possible to deduce the space group of the diffracting crystal. History of Electron Microscopy 113 Gjønnes, J. and Moodie, A.F., Extinction conditions in the dynamic theory of electron diffraction. Acta Cryst. 19 ~1965! 65–67. Howie, A. and Basinski, Z.S., Approximations of the dynamical theory of diffraction contrast. Phil. Mag. 17 ~1968! 1039–1063. 1966 A beautifully simple method of stacking fault determination, which does not require the use of dark-field images ~experimentally very difficult to produce in 1966!; instead, it relies on bright-field images together with the fine structure of the diffraction spots. 1968 Introduction of the absorption parameters necessary for quantitative interpretation of defect images showing diffraction contrast. Van Landuyt, J., Gevers, R. and Amelinckx, S., On the determination of the nature of stacking faults in f.c.c metals from the bright field image. Phys. Stat. Sol. 18 ~1966! 167–172. 1966 In six closely linked papers, the connection between diffraction fine structure associated with defects and bright and dark field imaging is made. The particularly clear interplay between the descriptive mathematics and the physical reality is very instructive. Gevers, R., Landuyt, J. van and Amelinckx, S., The fine structure of spots in electron diffraction resulting from the presence of planar interfaces and dislocations. Phys. Stat. Sol. 18 ~1966! 343–361 and 363–378; 26 ~1968! 577–590; Ridder, R. de, Landuyt, J. van, Gevers, R. and Amelinckx, S., 30 ~1968! 797–815; 38 ~1970! 747–756 and 797–815. 1967 The development of Kikuchi maps to permit direct visual determination of the foil orientation, without the need for complete indexing of diffraction patterns, makes the life of the experimental microscopist significantly easier. Okamoto, P.R., Levine, E. and Thomas, G., Kikuchi maps for hcp and bcc crystals. J. Appl. Phys. 38 ~1967! 289–296. 1968 A major improvement in the accuracy of conventional SAD pattern indexing. Ryder, P.L. and Pitsch, W., On the accuracy of orientation determination by selected area electron diffraction. Phil. Mag. 18 ~1968! 807–816. 1968 Critical examination of the two-beam approximation and the column approximation and development of a perturbation theory for the study of scattering is developed. The Howie–Whelan approach is extended to the many-beam case without the need for the column approximation. This heavily used paper is essential for the quantitative interpretation of diffraction-contrast images of defects. Humphreys C.J. and Hirsch P.B., Absorption parameters in electron diffraction theory. Phil. Mag. 18 ~1968! 115–122. 1968 The collector-plate mechanism, which accounts beautifully for the growth kinetics of grain-boundary allotriomorphs, is developed in this paper, in which the TEM is used to image the size and shape of the precipitates as a function of time and temperature. Aaron, H.B. and Aaronson, H.I. Growth of grain boundary precipitates in Al-4% Cu by interfacial diffusion, Acta Metall. 16 ~1968! 789–798. 1969 First conclusive demonstration of the capability of microstructural analysis in the TEM to distinguish the different mechanisms of formation of the precipitate-free zone ~PFZ!, a crucial microstructural defect that controls the properties of many precipitation-hardened engineering alloys. Unwin, P.N.T., Lorimer, G.W. and Nicholson, R.B., The origin of the grain boundary precipitate-free zone, Acta Metall. 17 ~1969! 1363–1377. 1969 The grain-boundary precipitation behavior in systems that form the basis of commercial high-strength Al alloys is revealed and explained. Unwin, P.N.T. and Nicholson, R.B., The nucleation and initial stages of growth of grain boundary precipitates in Al-Zn-Mg and Al-Mg alloys. Acta Metall. 17 ~1969! 1379–1393. 1969 In a landmark paper, it is demonstrated that lattice images of complex oxides could be interpreted directly in terms of projected crystal structure. This drew on the phase-contrast ideas of Heidenreich and also on the theoretical work of Cowley and Moodie. This and succeeding papers in the early 1970s by Allpress and coworkers transformed much of solidstate chemical thought of that time. Allpress, J.G., Sanders, J.V. and Wadsley, A.D. Multiple phase formation in the binary system Nb2O5–WO3 . 114 F. Haguenau et al. VI. Electron microscopic observation and evaluation of non-periodic shear structures. Acta Cryst. B25 ~1969! 1156–1164. clear that HREM images may be interpreted in this way, but only under very stringent experimental conditions. 1969 The first use of the weak-beam technique for the study of dislocations and so forth. The technique is now among the most widely used in materials science TEM. Cockayne, D.J.H., Parsons, J.R. and Hoelke, C.W., A study of the relationship between lattice fringes and lattice planes in electron microscope images of crystals containing defects. Phil. Mag. 24 ~1971! 139–153. Cockayne, D.J.H., Ray, I.L.F. and Whelan, M.J., Investigations of dislocation strain fields using weak beams. Phil. Mag. 20 ~1969! 1265–1270. 1973 The first use of spectroscopy in the TEM to measure low-temperature diffusion directly from inhomogeneous areas of thin foils. 1970 Demonstration that the growth of second-phase precipitates in Cu-Ni-Fe follows the predictions of Cahn’s theory of spinodal decomposition, completed only two years before. Doig, P. and Edington, J.W., Low temperature diffusion in Al-7-wt.% Mg and Al-4-wt.% Cu alloys. Phil. Mag. 28 ~1973! 961–970. Butler, E.P. and Thomas, G. Structure and properties of spinodally-decomposed Cu-Ni-Fe alloys, Acta Metall. 18 ~1970! 347–365. 1971 At the time when the validity of interpreting HREM images in terms of crystal structure was in serious doubt ~see Cockayne et al., 1971, below!, this brief letter shows an unambiguous correlation between lattice image contrast and projected crystal structure, for a complex oxide Ti2Nb10O29 . It vindicates the earlier work of Allpress et al. 1969 ~q.v.! and preceded image simulation techniques. Iijima, S., High-resolution electron microscopy of crystal lattice of titanium-niobium oxide. J. Appl. Phys. 42 ~1971! 5891–5893. 1971 Description of the use of inelastically scattered electrons to form dark-field images showing bright contrast corresponding to single atoms of thorium. This paper is now widely accepted as the first publication showing single atom contrast by transmission electron microscopy. Hashimoto, H., Kumao, A., Hino, K., Yotsumato, H. and Ono, A., Images of thorium atoms in transmission electron microscopy. Japan. J. Appl. Phys. 10 ~1971! 1115–1116. 1971 At a time when electron microscopes were becoming stable enough to produce high-resolution lattice images, this paper sounds a cautionary note on the dangers of interpreting “lattice fringes” intuitively in terms of lattice planes in defective crystals. It is now 1973 One of a series of papers in which high-resolution lattice images are used to investigate the initial stages of precipitation. Much of the analysis is later proved to be flawed because of the use of tilted illumination, and moreover, suitable image simulation techniques are not available at the time. Nevertheless, this work first shows the latent power of high resolution imaging in the TEM to reveal the smallest scale phenomena that ultimately provide strengthening mechanisms for most engineering materials. Philips, V.A., Lattice resolution measurement of strain fields at Guinier–Preston zones in Al-3.0% Cu. Acta Metall. 21 ~1973! 219–228. 1974 Allotropes of carbon distinguished by examining the shape of the near-edge fine structure in the EELS spectrum. Egerton, R. and Whelan, M.J., The electron energy loss spectrum and band structures of diamond. Phil. Mag. 30 ~1974! 739–749. 1975 Detailed description of the computational developments needed to produce simulations of diffraction contrast effects. Image matching methods could be employed to derive with considerable accuracy stacking fault parameters and other data from images. Humble, P. and Forwood, C.T., Identification of grain boundary dislocations. I. The simultaneous two-beam method for obtaining experimental and computed electron micrographs. II. Image matching using the simultaneous two-beam technique. Phil. Mag. 10 ~1975! 1011–1023 and @CTF and PH# 1025–1048. History of Electron Microscopy 115 1975 The definitive paper on quantitative energy-dispersive X-ray microanalysis from thin crystals—giving rise to the well known “Cliff–Lorimer factors.” cates. It illustrates the power of HREM to examine mineral systems that have undergone alteration in the earth’s crust to form new structures. Cliff, G. and Lorimer, G.W., The quantitative analysis of thin specimens. J. Microscopy 103 ~1975! 203–207. Veblen, D.R., Buseck, P.R. and Burnham, C.W., Asbestiform chain silicates: New minerals and structural groups. Science 198 ~1977! 359–365. 1976 First complete development of the theory of electron diffraction based on the wave-mechanical formulation, including the concept of dispersion surfaces and Bloch waves. Despite the title, the important topic of contrast from planar defects is also covered. Metherell, A.J.F., Diffraction of electrons by perfect crystals, in Electron Microscopy in Materials Science, Part II, pp. 397–552 ~U. Valdrè and E. Ruedl, eds; Commission of the European Communities, Brussels 1976!. 1977 First treatment of the fundamental aspects of convergent-beam electron diffraction, a technique that is in growing use for measurement of strain and symmetry as well as for structure factor determination in crystals. Jones, P.M., Rackham, G.M. and Steeds, J.W., Higher order Laue zone effects in electron diffraction and their use in lattice parameter determination. Proc. Roy. Soc. (London) A354 ~1977! 197–222. 1977 Introduction of the first quantitative methods for absorption correction in thin foils and the singlescattering approximation for spatial resolution determination, as well as the first attempt to calculate the Cliff–Lorimer k factors essential for thin films. Goldstein J.I., Costley, J.L., Lorimer, G.W. and Reed, S.J.B., Quantitative X-ray analysis in the electron microscope. Scanning Electron Microsc. ~1977! 315–324. 1977 The best example of the first method of quantitative EELS microanalysis. This paper reports plasmon-loss measurements that show the validity of Cahn’s model whereby solute redistribution controls the kinetics of grain boundary migration during precipitation. Porter, D.A. and Edington, J.W., Microanalysis and cell boundary velocity measurements for the cellular reaction in a Mg-9% Al alloy. Proc. Roy. Soc. (London) A358 ~1977! 335–350. 1977 HREM is used to elucidate new mineral structures, which form a bridge between chain and sheet sili- 1978 The first paper to show the ability of spectroscopy in the TEM to detect interfacial segregation at the subnanometer level. Doig, P. and Flewitt, P.E.J., The influence of temper embrittlement on the stress corrosion susceptibility of Fe-3 wt. % Ni alloys. Acta Metall. 26 ~1978! 1283– 1291. 1979 A new form of the the Z-contrast imaging technique is presaged. Also described is the ability of the STEM to select electrons for imaging in a much more versatile way than the TEM which was ~and still is! constrained by analogue aperture-selection techniques. Howie, A., Image contrast and localized signal selection techniques. J. Microscopy 117 ~1979! 11–23. 1979 A novel method for imaging thin ~below 1 nm! amorphous boundary layers using the diffuse-scattered electrons. Clarke, D.R., On the detection of thin intergranular films by electron microscopy. Ultramicroscopy 4 ~1979! 33–44. 1979 The first demonstration of the effect of orientationdependence on near-edge fine structure, showing the strong effect of bonding on EEL spectra. Leapman, R.D. and Silcox, J., Orientation dependence of core edges in electron-energy-loss spectra from anisotropic materials. Phys. Rev. Lett. 42 ~1979! 1361– 1364. 1979 This paper is the basis of quantitative ionization loss spectrometry providing the fundamental parameters to convert edge intensity into elemental concentration. Egerton, R.F., K-shell ionization cross-sections for use in microanalysis. Ultramicroscopy 4 ~1979! 169–179. 1980 The determination of binary and ternary phase diagrams at hitherto inaccessibly low temperatures is 116 F. Haguenau et al. made feasible by high spatial resolution microanalysis in the TEM. At such temperatures, conventional techniques are useless ~Ni atoms jump once every 10,000 years in Fe at 3008C!. Romig, A.D. and Goldstein, J.I., Determination of the Fe-Ni and Fe-Ni-P phase diagrams at low temperatures ~700–3008C!. Metall. Trans. 11A ~1980! 1151– 1159. 1980 A superb illustration of the periodic nature of grain boundary defect structures, using them as diffraction gratings to scatter electrons. Carter, C.B., Donald, A.M. and Sass, S.L., The study of grain boundary thickness using electron diffraction techniques. Phil. Mag. A 41 ~1980! 467–475. 1981 Highly localized crystallographic information obtained by exploiting small probes. Cowley, J.M. and Spence, J.C.H., Convergent beam electron micro-diffraction from small crystals. Ultramicroscopy 6 ~1981! 359–366. 1982 The article by Leapman et al. is not the first description of energy-loss near-edge structure, but the first study to show the feasibility of using ELNES to give quantitative bonding information and, ultimately, phase identification. Leapman, R.D., Grunes, L.A. and Fejes, P.L., Study of the L23 edges in the 3d transition metals and their oxides, by electron-energy-loss spectroscopy with comparisons to theory. Phys. Rev. B 26 ~1982! 614–635. 1982 In this and the following paper ~“Electron damage in Cu3Au”, pp. 717–721!, the use of the Birmingham 1 MeV ~AEI EM7! microscope is described in its application to order–disorder phenomena. A number of these instruments were in use in the UK between 1971 and the late 1980s. These papers are examples of the large output from these microscopes. Hameed, M.Z., Smallman, R.E. and Loretto, M.H., H.V.E.M. study of ordering and disordering in Cu3Au. Phil. Mag. A 46~1982! 707–716. 1982 The explanation of grain boundary grooving in thin foil specimens, a phenomenon that can both aid in the interpretation of boundary images and also cause serious errors when interpreting analytical data from the same boundaries. Goodhew, P.J. and Smith, D.A., Grooving at grain boundaries in thin films. Scripta Metall. 16 ~1982! 91–94. 1983 The development of inert gas bubbles in irradiated metals presented serious problems in the nuclear energy industry and a series of papers presented much systematic work on the problem. In this example, the preferential growth of helium bubbles at grain boundaries is described. Goodhew, P.J., Helium bubble nucleation at grain boundaries. Phil. Mag. A 48 ~1983! 965–986. 1983 A beautiful method for discerning the location of atoms on specific crystal sites through their interaction with different Bloch waves ~also the best acronym in the field of TEM!. Spence, J.C.H. and Taftø, J., ALCHEMI; a new technique for locating atoms in small crystals. J. Microscopy 130 ~1983! 147–154. 1983 Presentation of a “real-space” approach to the simulation of lattice images, contrasting with the more widely used multislice approach of Cowley and Moodie. Dyck, D. van, High-speed computation techniques for the simulation of high-resolution electronmicrographs. J. Microscopy 132 ~1983! 31–42. 1983 Recognition of the importance of electron beam alignment for obtaining meaningful lattice images close to the resolution limit. This paper shows how the comafree axis could be found experimentally, based on Fourier transforms of images of amorphous films. Coma-free alignment routines are now widely used in the most critical HREM studies. Smith, D.J., Saxton, W.O., O’Keefe, M.A., Wood, G.J. and Stobbs, W.M., The importance of beam alignment and crystal tilt in high-resolution electron microscopy. Ultramicroscopy 11 ~1983! 263–281. 1983 The source of crucial tables of symmetry data from CBED patterns which permit the straightforward determination of space group symmetry from crystals as small as about 100 nm, far below the resolution limit of traditional X-ray diffraction methods. Steeds, J.W. and Vincent, R., Use of high-symmetry zone axes in electron diffraction in determining crystal point and space groups. J. Appl. Cryst. 16 ~1983! 317–324. History of Electron Microscopy 117 1984 A classic example of using CBED patterns for 3-D crystal structure determination. formed in the initial stage of copper oxidation. Acta Cryst. B 41 ~1985! 219–225. Raghavan, M., Scanlon, J.C. and Steeds, J.W., Use of reciprocal lattice layer spacing in convergent beam electron diffraction analysis. Metall. Trans. 15A ~1984! 1299–1302. 1986 One of a number of classic papers by this group describing investigations of important classes of structures. 1984 A contested claim that structures of inorganic compounds can be derived directly from HREM images. The claim that structure factor phases are preserved in HREM images ~after image processing in many cases! has met with some skepticism, but the work does represent a significant contribution to HREM image interpretation methods. Hovmöller, S., Sjogren, A., Farrants, G., Sundberg, M. and Marinder, B.-O., Accurate atomic positions from electron microscopy. Nature 311 ~1984! 238–241. 1984 A classic review of the many techniques for studying magnetic domain structures in the TEM. Lanteri, V., Mitchell, T.E. and Heuer, A.H., The morphology of tetragonal precipitates in partially stabilized ZrO2 . J. Amer. Ceram. Soc. 69 ~1986! 564–569. 1987 The nature of the atomic structure of ledges on the interfaces of growing precipitates is revealed. This paper is a superb demonstration of the power of combined imaging, diffraction, and spectroscopy to address one of the long-standing problems in materials science: the atomic mechanisms of precipitate growth. Howe, J. M., Dahmen, U. and Gronsky, R., Atomic mechanisms of precipitate growth. Phil. Mag. A. 56 ~1987! 31–61. 1984 A key HREM study showing how contrast in AlAs/ GaAs layers could be enhanced by exploiting chemically sensitive structure factors for particular diffracted beams. 1987 This paper in Ultramicroscopy encapsulates much of Hobbs’ work on electron irradiation damage, particularly on halides and also in quartz. In view of its findings, it is perhaps rather surprising that we can obtain any images at all of many materials, where the atoms will have been moved from their equilibrium lattice sites many times! Much earlier work is listed in this short survey. See also Section 2 and the book chapter listed here. Petroff, P.M., Gossard, A.C. and Weigmann W., Structure of AlAs–GaAs interfaces grown on ~100! vicinal surfaces by molecular beam epitaxy. Appl. Phys. Lett. 45 ~1984! 620–622. Hobbs, L.W., Radiation effects in analysis by TEM. In Quantitative Electron Microscopy ~Chapman, J.N. and Craven, A.J., eds! 399–445 ~Scottish Universities Summer School in Physics, Edinburgh 1984!. 1984 Representative of a series of papers describing what have become known as “quasicrystals”: crystalline materials displaying icosahedral “symmetry,” a completely unexpected discovery. Hobbs, L.W., Electron-beam sensitivity in inorganic specimens. Ultramicroscopy 23 ~1987! 339–344. Chapman, J.N., The investigation of magnetic domainstructures in thin foils by electron microscopy. J. Phys. D: Appl. Phys. 17 ~1984! 623–630. Shechtman, D., Blech, I., Gratias, D. and Cahn, J.W., Metallic phase with long-range orientational order and no translational symmetry. Phys. Rev. Lett. 53 ~1984! 1951–1953. 1985 By using special HREM techniques, it was possible to elucidate new structural phases, including Cu64O, a hitherto unknown phase. Guan, R., Hashimoto, H. and Kuo, K.H., Electron microsopic study of the structure of metastable oxides 1987 The Chinese school led by K.H. Kuo made many discoveries concerning quasicrystals. This survey gives a good idea of their contribution. Kuo, K.H., Quasicrystals and noncrystallographic symmetry. J. Electron Microsc. Tech. 7 ~1987! 277–282. 1987 The first of a series of papers that show how it is possible to extract thickness data from X-ray spectra, ultimately permitting the quantification of spectra without the need for on-line thickness determination, which had hampered analysis for the preceding decade. 118 F. Haguenau et al. Horita, Z., Sano, T. and Nemoto, M., Simplification of X-ray absorption correction in thin-sample quantitative microanalysis. Ultramicroscopy 21 ~1987! 271– 276. 1988 A simple approach to translating the low-loss intensity in any EELS spectrum into an accurate and quantitative measure of the specimen thickness—the crucial parameter in any quantitative microanalysis in the TEM. Malis, T., Cheng, S.C. and Egerton, R.F., EELS logratio technique for specimen-thickness measurement in the TEM. J. Electron Microsc. Tech. 8 ~1988! 193–200. 1989 The first description of a technique that has transformed spectroscopy in the TEM into a totally new mode of imaging. Jeanguillaume, C. and Colliex, C., Spectrum–image— the next step in EELS digital acquisition and processing. Ultramicroscopy 28 ~1989! 252–257. 1989 A coherent description of the crucial relationship between the symmetry displayed in CBED patterns and the symmetry of the diffracting crystal. Tanaka, M., Symmetry analysis. J. Electron Microsc. Tech. 13 ~1989! 27–39. 1991 The development of energy-filtered electron diffraction patterns for the determination of the radial distribution function of amorphous materials. This quantitative method of RDF determination makes the TEM a viable ~and cheaper! alternative to the synchrotron for the study of the structure of amorphous materials. Cockayne, D., McKenzie, D. and Muller, D., Electron diffraction of amorphous thin films using PEELS. Microsc. Microanal. Microstruct. 2 ~1991! 359–366. 1991 Demonstration of the feasibility of single-atom identification in the TEM, the “holy grail” of microanalysis. Krivanek, O.L., Mory, C., Tencé, M. and Colliex, C., EELS quantification near the single-atom detection level. Microsc. Microanal. Microstruct. 2 ~1991! 257– 267. 1991 The discovery of carbon nanotubes. This work spawned an unprecedented interest in these structures and their possible applications. One of the most influential and frequently cited papers in the field. Iijima, S., Helical microtubules of graphitic carbon. Nature 354 ~1991! 56–58. 1992 Z-contrast transmission electron microscopy was the first fundamentally new TEM imaging technique to appear in more than 20 years ~since weak-beam imaging!. Its power was convincingly demonstrated by Pennycook and coworkers. Pennycook, S.J., Z-contrast transmission electronmicroscopy—direct atomic imaging of materials. Annu. Rev. Mater. Sci. 22 ~1992! 171–195. 1994 M.A. O’Keefe, author of widely used simulation programs, reports on most of the related programs that have been made available by their authors, indicating the relationships between various software routines, and also showing where intercomparisons or crosschecks for consistency have been run. O’Keefe, M.A., Interpretation of HRTEM images by image simulation; an introduction to theory and practice. Proceedings 52nd Annual Meeting Microscopy Society of America ~Bailey, G.W. and Garratt-Reed, A.J., eds.! pp. 394–395 ~San Francisco Press, San Francisco 1994!. 1994 Information not provided by the X-ray diffraction pattern is shown to be retrievable from the electron diffraction data. O’Keefe, M.A. and Spence, J.C.H., On the average Coulomb potential and constraints on the electron density in crystals. Acta. Cryst. A 50 ~1994! 33–45. 1994 Result of a “round robin” series of tests carried out by various authors to provide a direct comparison of a number of simulation programs. It gives an objective review of the relative merits of the different suites. Van Dyck, D. and Op de Beek, M., Comparison and test of HRTEM image simulation programs. Ultramicroscopy 55 ~1994! 435–457. 1994 Sinclair has produced many significant papers describing in situ studies of crystallization, boundary migration, and so forth. This example illustrates the power of the technique and its ability to produce fresh insights into solid-state processes. History of Electron Microscopy 119 Gutekunst, G., Mayer, J. and Rühle, M., The niobium sapphire interface—structural studies by HREM, Scripta Metall. 31 ~1994! 1097–1102. thin sections of whole tissue biopsies were required, fixation was followed by dehydration, embedding, and staining. The remarkable qualities of osmium tetroxide as a fixative had long been known, and it had already been employed by Ladislaus Marton in 1934 and by Carl Wolpers and Helmut Ruska in 1939. In 1945, Keith Porter, A. Claude, and E.F. Fullam used OsO 4 for fixation and staining of cells in tissue culture ~see Section 4.2!. George Palade subsequently recommended the use of buffered OsO 4 , stressing the relevance of the pH and osmolarity for the preservation of cell and tissue ultrastructure. 1996 A rigorous mathematical approach to analysing EDX spectra for improved quantification and sensitivity. 1952 Palade, G.F., A study of fixation for electron microscopy. J. Exp. Med. 95 ~1952! 285–298. Titchmarsh, J.M. and Dumbill, S., Multivariate statistical analysis of FEG–STEM EDX spectra. J. Microscopy 184 ~1996! 195–207. 1956 Potassium permanganate fixation is introduced by J.H. Luft. This technique was particularly useful for high resolution studies of biological membranes. Sinclair, R. and Konno, T.J., In-situ HREM— application to metal-mediated crystallization. Ultramicroscopy 56 ~1994! 225–232. 1994 A very detailed and quantitative interpretation of HREM images of a heterointerface in which all cation positions are accurately determined by critical matching of experimental and simulated images. 1997 The first ab initio structure determination of nanoparticle structure in a matrix using only electron optical methods. A rough model was derived from exit waves reconstructed from HREM images. Refinement to an R value of 3.1% was achieved, with dynamical scattering being taken into account. Zandbergen, H.W., Andersen, S.J. and Jansen, J., Structure determination of Mg 5Si6 particles in Al by dynamic electron diffraction studies. Science 277 ~1997! 1221–1225. 4. E LECTR ON M ICR OSCOPY L IFE S CIENCES IN THE 4.1. Techniques Devised for the Study of Biological Material Chemical Fixation The progress in electron microscope design in the 1940s could not be used effectively in the life sciences without tackling the problem of sample preparation. The first and indispensable requirement was the quality of preservation. In the early days, different procedures were developed in parallel, depending on whether spread cells or tissue biopsies were to be examined. In the first case, cells were simply grown on lamellae coated with formvar or collodion. The resulting film was detached and its thinnest areas were deposited over a grid and exposed to osmium vapor. When Luft, J.H., Permanganate—a new fixative for electron microscopy. J. Biophys. Biochem. Cytol. 2 ~1956! 799–801. Mollenhauer, H.H., Permanganate fixation of plant cells. J. Biophys. Biochem. Cytol. 6 ~1959! 431–436. 1963 Use of aldehyde for fixation. Sabatini, D.D., Bensch, K. and Barrnett, R.J., Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17 ~1963! 19–58; Karnovsky, M.J., A formaldehyde–glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27 ~1965! 137A. The Use of Low Temperature as a Means of Fixation Besides these chemical approaches, new physical methods for specimen preservation at very low temperatures ~freezedrying, freeze-fracturing, freeze-etching! were developed. These specimen freezing techniques were introduced in 1946 by Ralph Wyckoff and the use of critical-point phenomena followed soon after. Wyckoff, R.W.G., Frozen-dried preparation for the electron microscope. Science 104 ~1946! 36–37; Anderson, T.F., The use of critical point phenomena in preparing specimens for the electron microscope. J. Appl. Phys. 21 ~1950! 724. 1951 Low-temperature electron microscopy was used to study biological membranes by Humberto Fernández-Morán. 120 F. Haguenau et al. Fernández-Morán, H., The fine structure of vertebrate and invertebrate photoreceptors as revealed by lowtemperature electron microscopy. In The Structure of the Eye ~Smelser, C., ed.! 521–530 ~Academic Press New York 1951!. 1952 An improved procedure for freeze-drying was introduced by Robley Williams. Williams, R.C., A method of freeze-drying for electron microscopy. Exp. Cell Res. 4 ~1952! 188–201; Sjöstrand, F.S. and Baker, R.F., Fixation by freeze-drying for electron microscopy of tissue cells. J. Ultrastruct. Res. 1 ~1958! 239–246. 1966 The mechanism of freeze-fracture of biological membranes was discovered by David Branton, who provided the evidence that, at low temperature, membranes split along the hydrophobic core. Branton, D., Fracture faces of frozen membranes. Proc. Natl. Acad. Sci. USA 55 ~1966! 1048–1056. 1982 Technological advance was accomplished by J. Escaig, who developed an apparatus for rapid freezing of biological objects. Escaig, J., New instruments which facilitate rapid freezing at 83K and 6K. J. Microscopy 126 ~1982! 221–229. Embedding Media 1949 Introduction of methacrylate as an embedding medium for biological specimens to be cut by a microtome. Newman, S.B., Borysko, E. and Swerdlow, M., New sectioning techniques for light and electron microscopy. Science 110 ~1949! 66–68. 1956 Development of new resins that do not exhibit polymerization defects. Kellenberger, E., Schwab, W. and Ryter, A., L’utilisation d’un co-polymère du groupe des polyesters comme matériel d’inclusion en ultramicrotomie. Experientia 12 ~1956! 421–422 @polyester resins, Vestopal#. Glauert, A.M. and Glauert, R.H., Araldite as an embedding medium for electron microscopy. J. Biophys. Biochem. Cytol. 4 ~1958! 191–194. Luft, J.H., Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9 ~1961! 409– 414. 1960 Introduction of water-soluble embedding media in order to circumvent the deleterious dehydration stages in ethanol. Staübli, W., Nouvelle matière d’inclusion hydrosoluble pour la cytologie électronique. C.R. Acad. Sci. Paris 250 ~1960! 1137–1139; Leduc, E. and Bernhard, W., Essais de cytochimie ultrastructurale. Action sur la chromatine. C.R. Acad. Sci. Paris 250 ~1960! 2948– 2950. 1982 Introduction of immunology. an important technique for Carlemalm, E., Garavito, R.M. and Villiger, W., Resin development for electron microscopy and an analysis of embedding at low temperature. J. Microscopy 126 ~1982! 123–143. Thin Sectioning of Embedded Material and the Development of Ultramicrotomy 1943 An early ultramicrotome with thermal advance is developed by Fritiof Sjöstrand in the Karolinska Institutet, Stockholm and used to study skeletal muscle. Sjöstrand, F.S., Eine neue Methode zur Herstellung sehr dünner Objektschnitte für die elektronenmikroskopische Untersuchung von Geweben nebst einigen vorläufigen elektronenmikroskopischen Beobachtungen über den submikroskopischen Bau der Skelettemuskelfaser. Ark. Zool. 35A ~1943! No. 5, 18 pp. 1948 From this date onwards, ultramicrotomy developed rapidly. Pease, D.C. and Baker, R.F., Sectioning techniques for electron microscopy using a conventional microtome. Proc. Soc. Exp. Biol. Med. 67 ~1948! 470–474; Bretschneider, L.H., A simple technique for the electron microscopy of cell and tissue sections. Proc. Kon. Ned. Acad. Wetenschappen 52 ~1949! 654–666; Hillier, J., Refined sectioning techniques for the electron microscope. C.R. du 1er Congrès International de Microscopie Electronique, Paris 1950, vol. 1, pp. 592–599 ~Editions de la Revue d’Optique, Paris 1953!; Latta, H. and Hartmann, F.J., Use of a glass edge in thin sectioning for electron microscopy. Proc. Soc. Exp. Biol. Med. 74 ~1950! 436–439; Porter, K.R. and Blum, J., A study of microtomy for electron microscopy. Anat. Rec. 117 History of Electron Microscopy ~1953! 685–710; Sjöstrand, F.S., A new microtome for ultrathin sectioning for high resolution electron microscopy; Exp. Cell Res. 9 ~1953! 114–115; FernándezMorán, H. Applications of a diamond knife for ultrathin sectioning to the study of the fine structure of biological tissues and metals. J. Biophys. Biochem. Cytol. 2 ~1956! Supplement 4, 29–31; Tokuyasu, K.T., A technique for ultra cryotomy of cell suspensions and tissues. J. Cell Biol. 57 ~1973! 551–565 ~a pioneering work, subsequently applied extensively in ultrastructural cytochemistry!. Staining and Contrast Enhancement Techniques One of the problems raised by specimen examination was the difficulty of distinguishing the object itself from its surrounding support. Contrast enhancement was a necessity. Gibbons, I.R. and Bradfield, J.R.G., Experiments on staining thin-sections for electron microscopy. Proc. EUREM-1 ~Stockholm, 1956! 121–124. In 1955, Cecil Hall published a study of the effect of various staining methods on viruses, and described viral particles that were not positively stained by phosphotungstic acid ~PTA! but appeared to be embedded in the dried reagent and displayed a negative contrast. A similar observation was made by Hugh Huxley in 1956 in his studies of tobacco mosaic virus ~TMV!. Sidney Brenner and Robert Horne subsequently gave a full explanation of the relevance of this negative staining for the study of viruses and macromolecules. A comprehensive review of negative staining and other methods of visualizing viruses in the electron microscope was written soon after by R.C. Valentine. Hall, C.E., Electron densitometry of stained virus particles. J. Biophys. Biochem. Cytol. 1 ~1955! 1–12. Watson, M.L., Staining of tissue sections for electron microscopy with heavy metals. II. Application of solutions containing lead and barium. J. Biophys. Biochem. Cytol. 4 ~1958! 724–730. Brenner, S. and Horne, R.W., A negative staining technique for high resolution electron microscopy of viruses. Biochem. Biophys. Acta 34 ~1959! 103–110. Valentine, R.C., Contrast enhancement in the electron microscopy of viruses. Adv. Virus Res. 8 ~1961! 287– 318. 121 Reynolds, E.S., The use of lead citrate at high pH as electron opaque stain in electron microscopy. J. Cell Biol. 17 ~1963! 208–212. 1969 Description of a regressive staining technique for distinguishing between DNA- and RNA-carrying structures in the nucleus. This method allowed the perichromatin fibril structures to be described for the first time; these correspond in situ to RNA protein complexes. Bernhard, W., A new staining procedure for electron microscopical cytology. J. Ultrastruct. Res. 27 ~1969! 250–265. Metal Shadowing and Replicas The introduction of metal shadowing by H.O. Müller in 1942 has been mentioned in Section 2; Williams and Wyckoff independently combined freeze-drying and metal shadowing for the electron microscope study of viruses and macromolecules. Williams, R.C. and Wyckoff, R.W.G., Electron shadow microscopy of virus particles. Proc. Soc. Exp. Biol. Med. 58 ~1945! 265–270. 1950 Development of methods for preparing replicas of frozen material under vacuum. Hall, C.E., Low-temperature replica method for electron microscopy. J. Appl. Phys. 21 ~1950! 61–62. 1957 The replica technique was adapted to permit frozen samples to be fractured and etched before deposition of the replica layer. Steere, R.L., Electron microscopy of structural detail in frozen biological specimens. J. Biophys. Biochem. Cytol. 3 ~1957! 45–60. Immunolabeling, Immunochemistry The use of electron-dense ferritin coupled with immunoglobins for the identification of specific antigen sites in the electron microscope was introduced in 1959 by S.J. Singer. This method was extensively applied in virology by C. Morgan and coworkers soon afterwards ~see the comprehensive review by Andres et al., 1967!. Singer, S.J., Preparation of an electron-dense antibody conjugate. Nature 183 ~1959! 1523–1524. 122 F. Haguenau et al. Andres, G.A., Hsu, K.C. and Seegal, B.C., Immunoferritin technique for identification of antigens by electron microscopy. In Handbook of Experimental Immunology ~Weis, D.M., ed.! 527–570 ~Blackwell, Oxford 1967!. From 1966 on, new methods of antibody labelling were devised. Immuno-enzymatic techniques were employed before embedding while immunogold methods were applied to tissue sections after embedding or after ultracryotomy. Immunoenzymatic labeling In 1966, Graham and Karnovsky developed a cytochemical method of localizing horseradish peroxidase ~HRP!. It is extensively used for the ultrastructural immunocytochemistry of peroxidase-labeled antibodies. Graham, R.C. and Karnovsky, M.J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14 ~1966! 291–302. Avrameas, S. and Uriel, J., Méthode de marquage d’antigènes et d’anticorps avec des enzymes et son application en immunodiffusion. C.R. Acad. Sci. Paris D262 ~1966! 2543–2545. Nakane, P.K. and Pierce, G.B, Enzyme-labeled antibodies: preparation and application for the localization of antigens. J. Histochem. Cytochem. 14 ~1966! 929–931. Tougard, C., Picart, R., Tixier-Vidal, A., Kerdelhué, B. and Jutisz, M., In situ immunochemical staining of gonadotropic cells in primary cultures of rat anterior pituitary cells with the peroxidase labelled antibody technique. In Proc. 2nd Int. Symp. on Electron Microscopy and Cytochemistry ~Wisse, E., Daems, W., Molenar, J. and van Duijn, P., eds! 163–166 ~NorthHolland, Amsterdam 1974!. Description of an original method allowing photonic and electron localization of antigens and enzymes in tissue culture and in situ. Geuze, H.J., Slot, J.W., van der Ley, P.A., Scheffer, R.C. and Griffith, J.M., Use of colloidal particles in doublelabeling immunoelectron microscopy of ultrathin frozen tissue sections. J. Cell Biol. 89 ~1981! 653–665. Keller, G.A., Tokuyasu, K.T., Dutton, A.H. and Singer, S.J., An improved procedure for immunoelectron microscopy: ultrathin plastic embedding of immuno- labeled ultrathin frozen sections. Proc. Natl. Acad. Sci. USA 81 ~1984! 5744–5747. Immunogold labeling Romano, E.L. and Romano, M., Staphylococcal protein A bound to colloidal gold: A useful reagent to label antigen–antibody sites in electron microscopy. Immunochemistry 14 ~1977! 711–720. Bendayan, M., Roth, J., Perrelet, A. and Orci, L., Quantitative immunocytochemical localization of pancreatic proteins in subcellular compartments of the rat acinar cells. J. Histochem. Cytochem. 28 ~1980! 149–160. de Mey, J., The preparation of immunoglobulin gold conjugates ~IGS! reagents and their use as markers for light and electron microscopy immunocytochemistry. In Immunocytochemistry ~Cuello, A.E., ed.!, pp. 347– 372 ~Wiley, Chichester 1983!. High-Resolution Autoradiography 1956 First attempts to use the electron microscope for autoradiography by Liquier–Milward and others. Liquier-Milward, J., Electron microscopy and radioautography as coupled techniques in tracer experiments. Nature 177 ~1956! 619; O’Brien, R.T. and George, L.A., Preparation of autoradiograms for electron microscopy. Nature 183 ~1959! 1461–1462; Pelc, S.R., Coombes, J.D. and Budd, G.C., On the adaptation of autoradiographic techniques for use with the electron microscope. Exp. Cell. Res. 24 ~1961! 192– 195; Przybylski, R.J., Electron microscope autoradiography. Exp. Cell. Res. 24 ~1961! 181–184; Caro, L.G. and Tubergen, R.P. van, High resolution autoradiography. I. Methods, and Caro, L.G., II. The problem of resolution. J. Cell Biol. 15 ~1962! 173–199; Granboulan, P., Granboulan, N. and Bernhard, W., Application de l’autoradiographie à la microscopie électronique. J. Microscopie 1 ~1962! 75–80. The Lausanne group headed by B. Droz played a pioneering role in the neurobiological domain. Young, A.W. and Droz, B., The renewal of protein in retinal rods and cones. J. Cell Biol. 39 ~1968! 162–184. Osborne, M.J., Droz, B., Meyer, P. and Morel, F., Angiotensin II: Renal localisation in glomerular mesiangial cells by autoradiography. Kidney Int. 8 ~1975! 245–256. History of Electron Microscopy For a comprehensive review, see Salpeter, M.M., General area of autoradiography at the electron microscope level. In Methods in Cell Physiology ~Prescott, D., ed.! vol. 2, 229–253 ~Academic Press, New York 1966!. Techniques in Molecular Biology The basic protein film technique for spreading DNA molecules was introduced as early as 1959 by Kleinschmidt et al., but it was only about 10 years later that the development of genetic engineering enabled electron microscopy to play an important role in the molecular biology of eukaryote genes. The heteroduplex DNA–DNA method disclosed regions of homology shared by related genomes. The study of DNA–protein complexes by Dubochet’s method showed that DNA was associated with histones in the same configuration as that of cellular chromatin. Finally the DNA–RNA heteroduplex method ~DNA– RNA hybridization and R-loops! unseated the dogma of the gene–protein colinearity ~well established for bacterial chromatin! by unraveling the splicing phenomenon. Kleinschmidt, A. and Zang, R.K., Über Desoxyribonucleinsäure-Molekeln in Protein-Mischfilmen. Z. Naturforsch. 14b ~1959! 770–779. Davis, R. and Davidson, N., Electron-microscopic visualization of deletion mutations. Proc. Natl. Acad. Sci. USA 60 ~1968! 243–250. Dubochet, J., Ducommun, M., Zollinger, M. and Kellenberger, E., A new preparation method for darkfield electron microscopy of biomacromolecules. J. Ultrastruct. Res. 35 ~1971! 147–167. Miller, O.L. and Bakken, A.H., Morphological studies of transcription. Acta Endocrinol. 168 ~1972! Suppl., 155–177. 4.2. The Normal Cell and Its Organelles The detail that became visible with the passage from a resolution of the order of micrometers ~light microscope! to a few hundreds of ångström units with the electron microscope was a revelation. The fine structure of the cell and its organelles were seen for the first time and it became imperative to interpret these structures in terms of their function. To this end, it was important that these elements be isolated. This was made possible by the development of the techniques of cellular fractionation combined with differen- 123 tial centrifugation. The way to an integrated view of the cell was now open. The pioneers in this domain were Albert Claude and the Rockefeller group with George E. Palade, to whom the Nobel Prize was awarded in 1974, together with Christian de Duve from Louvain, Belgium. Other groups at the N.I.H. National Cancer Institute ~Bethesda, MD! and in Europe also made basic contributions. As early as 1938, A. Claude with Joseph Bloom at the Rockefeller Institute constructed the first refrigerated ultracentrifuge. He was thus able to show the stratification of the various cellular organelles into superposed layers. He coined the term “microsome” to designate the layer that contains the polysome ~ribosomes, RNA, and endoplasmic reticulum!. Claude, A., Studies on cells. Morphology, chemical constitution and distribution of biological functions. Harvey Lectures 43 ~1948! 121–164. A general survey. Kuff, E.L., Hogeboom, G.H. and Dalton, A.J., Centrifugal, biochemical and electron microscope analysis of cytoplasmic particulates in liver homogenates. J. Biophys. Biochem. Cytol. 2 ~1956! 33–53. Palade, G.E. and Siekevitz, P., On liver microsomes. An integrated morphological and biochemical study. J. Biophys. Biochem. Cytol. 2 ~1956! 171–198. ~Other references to the various fractionation studies are listed under the corresponding organelle.! The Cell Membrane (Including Junctional Complexes) The outer surface of the cell is limited by a trilaminar structure: the unit membrane ~Robertson!. Specialized sites of firm attachment between adjacent cells in epithelia comprise tight and intermediate junctions as well as desmosomes. Bennet, H.S., The concept of membranous flow and membrane vesiculation as mechanisms of active transport and ion pumping. J. Biophys. Biochem. Cytol. Suppl. 2 ~1956! 99–103. Neville, D.M., The isolation of a cell membrane fraction from rat liver. J. Biophys. Biochem. Cytol. 8 ~1960! 413–422. Stein, W.D., Intra-protein interaction across a fluid membrane as a model for biological transport. In Membrane Proteins, Proc. Symp. Sponsored by the New York Heart Association and Little Brown Co., Boston ~1969! pp. 81–88. 124 F. Haguenau et al. Singer, S.J. and Nicolson, L., The fluid mosaic model of the structure of cell membranes. Science 175, ~1972! 720–731. The junctional complex In the mid-1960s, J.D. Robertson used permanganate fixation of club-endings in cell synapses to demonstrate the presence of a subunit pattern, thought to be associated with junctional membranes. Robertson, J.D., The occurrence of a subunit pattern in the unit membrane of club endings in Mauthner cell synapses in goldfish brains. J. Cell Biol. 19 ~1963! 201–221. Soon after, E.L. Benedetti and P. Emmelot found a hexagonal array of particles in negatively stained liver plasma membrane preparation; they were later found to be junctional domains that could be purified, being insoluble in detergent. This finding opened a new avenue for the biochemical characterization of junctional constituents. Benedetti, E.L. and Emmelot, P., Electron microscopic observations on negatively stained plasma membranes isolated from rat liver. J. Cell Biol. 26 ~1965! 166–174. In the same period, J.P. Revel and M.J. Karnovsky independently revealed that the minute gap between the closely adjoining plasma membranes of neighboring cells comprised the hexagonal subunit pattern. This membrane– membrane structure had previously been known as a “gap junction.” Revel, J.P. and Karnovsky, M.J., Hexagonal array of subunits in intercellular junctions of mouse heart and liver. J. Cell Biol. 33 ~1967! C7–C12. Important studies on the structure and function of gap junctions and other types of junctional domains were made by N.B. Gilula. Unger, V., Kumar, N., Gilula, N.B. and Yaeger, B., Threedimensional structure of a recombinant gap junction membrane channel. Science 283 ~1999! 1176–1179. For a review, see Franke, W.W., Cowin, P., Schmelz, M. and Kapprell, H.-P., The desmosomal plaque and the cytoskeleton. CIBA Foundation Symp. 125 ~1987! 26–48. The Nucleus: Chromatin, Nucleolus The difficulty in exploring the nucleus ~as opposed to the cytoplasm! in the beginnings of electron microscopy resided in the packaging of an enormous quantity of chromatin and ribonucleoproteins and the absence of internal membranes isolating the individual components, except for the nucleolus or the nuclear bodies. The first nuclear organelle to be described was the nucleolus. Bernhard, W., Haguenau, F. and Oberling, C., L’ultrastructure du nucléole de quelques cellules animales revelée par le microscope électronique. Experientia 8 ~1952! 58–69. @This was the first demonstration of the existence of a “filamentous” inward configuration after Estable and Sotelo had described it with the phase-contrast microscope in 1950.# Later, the role of the nucleolus in transcription was illustrated, and gene transcripts ~perichromatin fibrils and interchromatin granules! were described. Monneron, A. and Bernhard, W., Fine structural organization of the interphase nucleus in some mammalian cells. J. Ultrastruct. Res. 27 ~1969! 266–288. @Description of gene transcripts ~perichromatin fibrils! for the first time in situ.# Puvion, E. and Puvion-Dutilleul, F., Ultrastructure of the nucleus in relation to transcription and splicing; roles of perichromatin fibrils and interchromatin granules. Exp. Cell Res. 229 ~1996! 217–225. The molecular spreading technique made it possible to visualize nucleolar ~as well as nonnucleolar! genes. Miller, O.L. and Beatty, B.R., Visualization of nucleolar genes. Science 164 ~1969! 955–957. @The molecular spreading technique also enabled nonnucleolar genes to be visualized in transcription.# One of the earliest and major investigators was Hans Ris, who managed to unravel the intricate tangle of the chromatin and chromosome network of the animal nucleus as early as 1959. Further progress required the development of ultrastructural cytochemistry, well illustrated by W. Bernhard in Villejuif, the advent of the negative contrast technique and that of the molecular biology era. Ris, H., Ultrastructure and molecular organization of genetic systems. Can. J. Genet. Cytol. 3 ~1961! 95–120. History of Electron Microscopy @First description of the elementary chromatin fiber, 1.30 nm, in situ.# Ris, H., Ultrastructure of the animal chromosome. In Regulation of Nucleic Acid and Protein Biosynthesis ~Koningsberger, V.V. and Bosch, L., eds! 11–21 ~Elsevier, Amsterdam, London & New York 1967!. Olins, A., Carlson, R.D. and Olins, D.E., Visualization of chromosome structures. J. Cell Biol. 64 ~1975! 528–537. In the midst of this entangled meshwork of the so-called “loose” and “dense” chromatin and besides the nucleolus, other particulate structures were recognized, notably the nuclear bodies. De Thé, G., Rivière, M. and Bernhard, W., Examen au microscope électronique de la tumeur VX du lapin domestique et du papilloma de Shope. Bull. Assoc. Franç. Etude Cancer 47 ~1960! 570–584. Bouteille, M., Kalifat, S.R. and Delarue, J., Ultrastructural variations of nuclear bodies in human disease. J. Ultrastruct. Res. 19 ~1967! 474–486. The Nuclear Envelope The boundary that separates the nucleus from the ground cytoplasm is an extension of the endoplasmic reticulum. Note that the first two references here are not concerned with the mammalian cell. Callan, H.G. and Tomlin, S.G., Experimental studies on amphibian oocyte nuclei. I. Investigation of the structure of the nuclear membrane by means of the electron microscope. Proc. Roy. Soc. London B137 ~1950! 367– 378. @This work initiated studies on the subject.# Afzelius, R.A., The ultrastructure of the nuclear membrane of the sea urchin oocyte as studied with the electron microscope. Exp. Cell Res. 8 ~1955! 147–158. Watson, M.L., Further observations on the nuclear envelope of the animal cell. J. Biophys. Biochem. Cytol. 6 ~1959! 147–156. Franke, W.W., Nuclear envelope: structure and biochemistry of the nuclear envelope. Phil. Trans. Roy. Soc. London B268 ~1974! 67–93. The Centriole This is a minute body present in the cell center or centrosome. Its remarkable configuration was revealed by the electron microscope. 125 de Harven, E. and Bernhard, W., Étude au microscope électronique de l’ultrastructure du centriole chez les vertébrés. Z. Zellforsch. Mikrosk. Anat. 45 ~1956! 378– 398. ~One of the first descriptions in the vertebrates of its nine parallel tubules.! Amano, S., The structure of the centriole and spindle body as observed under the electron and phase contrast microscopes. Cytologia 22 ~1957! 193–212. Bessis, M. and Breton–Gorius, J., Les centrioles des cellules du sang, étude à l’état vivant et au microscope électronique. Bull. Microsc. Appl. 7 ~1957! 54–56. Yamada, E., Observation on the fine structure of centrioles in the mitotic cell. Kurume Med. J. 5 ~1958! 36–44. Cytoskeleton The architectural organization of the ground cytoplasm is made up of three components: microtubules ~30 nm!, microfilaments ~actin filaments, 9.5 nm!, and intermediate filaments ~7.12 nm!. Microtubules and microfilaments are also constituents of the centriole and are involved in protoplasmic movement, cell division, and contraction; they occur throughout the animal and plant realms. The reader is referred to Section 4.5 on plants and to the Atlas of Fine Structure compiled by D.W. Fawcett, listed in Section 6. The Endoplasmic Reticulum (Ergastoplasm) Studies on the endoplasmic reticulum provided the first evidence of the existence of a membrane network ~reticulum! running throughout the ground cytoplasm. Its specialized areas lined with ribosomes correspond to the ergastoplasm, which had been described long before by C. Garnier ~1890–1891!, who had coined the term from the Greek: to elaborate and transform. Two teams were predominant in the field and had considerable influence, attracting researchers worldwide: the Rockefeller group in New York with K.R. Porter and G.E. Palade and many famous collaborators and the Villejuif group around C. Oberling and W. Bernhard and numerous illustrious coworkers. Porter, K.R., Claude, A. and Fullam, E.F., A study of tissue culture cells by electron microscopy. J. Exp. Med. 81 ~1945! 233–246. @The first examination using electron microscopy of spread cells originating from tissue culture. The authors noticed the presence of a lace-like reticulum in the cytoplasm consisting of tubules, strands, and trabeculae.# 126 F. Haguenau et al. Dalton, A.J., Kahler, H., Striebich, M. and Lloyd, B., Fine structure of the hepatic interstitial and renal cells in the mouse as revealed by the electron microscope. J. Natl. Cancer Inst. 11 ~1950! 439–461. @First correct interpretation of the submicroscopic fibrils revealed by these early micrographs as representing the basophilic substance visualized in stained sections.# Bernhard, W., Haguenau, F., Gautier, A. and Oberling, C., La structure submicroscopique des éléments basophiles cytoplasmiques dans le foie, le pancréas et les glandes salivaires. Z. Zellforsch. 37 ~1952! 281–300. @This study, preceded by a Note à l’Académie des Sciences de Paris, is one of the earliest identifications of the ergastoplasm in the electron microscope.# Weiss, J., The ergastoplasm. J. Exp. Med. 98 ~1953! 607–618. @An excellent early and independent study by an American author who was aware of the work of C. Garnier.# Palade, G.E. and Porter, K.R., Studies on the endoplasmic reticulum. Its identification in situ. J. Exp. Med. 100 ~1954! 641–656. Palade, G.E., A small particulate component of the cytoplasm. J. Biophys. Biochem. Cytol. 1 ~1955! 59–87. Slayter, H.S., Warner, J.R., Rich, A. and Hall, C.E., The visualization of polyribosomal structure. J. Mol. Biol. 7 ~1963! 652–658. Louvard, D., Reggio, H. and Warren, G., Specific antigens localized in membranes of the endoplasmic reticulum and the Golgi. J. Cell Biol. 92 ~1982! 92–107. @First localization of intracellular membrane antigens.# Golgi Apparatus, Lysosomes (see also “Secretory Cells” below) This juxta-nuclear zone corresponds to the sites at which synthesis of glycoproteins occurs in the course of the protein transport throughout the cell. Lysosomes represent the sites at which the intracellular and extracellular materials are being degraded. They were identified in liver homogenates by C. de Duve ~1955! by their enzymatic properties and were later shown morphologically in the electron microscope by Alex Novikoff. The American leader in the field of Golgi studies, A.J. Dalton, with his collaborator Maria Felix ~Bethesda, MD! published basic electron microscope studies, which established unequivocally the reality of this structure, the existence of which had long been questioned and disputed. Dalton, A.J., Observation of the Golgi substance with the electron microscope. Nature 168 ~1951! 244–246. Haguenau, F. and Bernhard, W., L’appareil de Golgi dans les cellules normales et cancéreuses des vertébrés. Rappel historique et étude au microscope électronique. Arch. Anat. Microsc. Morphol. Exp. 44 ~1955! 27–55 @a review article#. Grassé, P.-P., Carasso, N. and Favard, P., Les dictyosomes ~appareil de Golgi! et leur ultrastructure. C. R. Acad. Sci. Paris 241 ~1955! 1243. @The universal distribution in the animal realm is shown here in molluscs and batrachians.# Morré, D.J., Hamilton, R.L., Mollenhauer, H.H., Mahley, R.W. Cunningham, W.P., Cheetham, R.D. and Lequire, V.S., Isolation of a Golgi apparatus-rich fraction from rat liver. I: Method and morphology. J. Biophys. Biochem. Cytol. 44 ~1970! 484–491. Novikoff, P.M., Novikoff, A.B., Quintana, N. and Hauw, J.-J., Golgi apparatus, GERL, and lysosomes of neurons in rat dorsal root ganglia, studied by thick section and thin section cytochemistry. J. Cell Biol. 50 ~1971! 859– 886. @In the term GERL, the G indicates the proximity to the Golgi apparatus at the trans face, ER corresponds to an area of the smooth Endoplasmic Reticulum that is considered to give rise to a variety of Lysosomes.# Rambourg, A., Marraud, A. and Chrétien, M., Tridimensional structure of the forming face of the Golgi apparatus as seen in the high voltage electron microscope after osmium impregnation of the small nerve cells in the semilunar ganglion of the trigeminal nerve. J. Microscopy 97 ~1973! 49–57. Orci, L., Glick, B.S. and Rothman, J.E., A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack. Cell 46 ~1986! 171–184. Mitochondria These are characteristic thread-like and rod-shaped organelles found in almost all cells. The inner folds of their external membranes provide a remarkable increase in surface area, allowing respiratory exchanges. History of Electron Microscopy Hogeboom, G.H., Schneider, W.C. and Palade, G.E., Cytochemical studies of mammalian tissues: I. Isolation of intact mitochondia from rat liver. J. Biol. Chem. 172 ~1948! 619–635. @Introduction of sucrose as a medium in fractionation procedures, which ensured good morphological preservation for the first time.# Palade, G.F., The fine structure of mitochondria. Anat. Rec. 114 ~1952! 427–452. @The first pictures revealing the remarkable inner folds, the role of which is to increase respiratory exchanges.# Sjöstrand, F.S., Electron microscopy of mitochondria and cytoplasmic double membranes. Nature 171 ~1953! 30–32. 4.3. Some Specialized Cells and Cell Structures We have so far dealt with the ultrastructural description of various cell organelles. A few examples of some specialized types of cells and constituents are now presented. They have been selected from an enormous literature and are, perforce, far from exhaustive. 127 Blood Cells, Vascular System Bessis, M., and Breton-Gorius, J., Iron particles in normal erythroblasts and normal and pathological erythrocytes. J. Biophys. Biochem. Cytol. 3 ~1957! 503–504. @The process of rhopheocytosis, the penetration of iron in the course of erythroblastic maturation, is described, a first example of endocytosis.# Rhodin, J.A.G., The ultrastructure of mammalian arterioles and precapillary sphincters. J. Ultrastruct. Res. 18 ~1967! 181–223. Behnke, O., An electron microscopic study of the megacaryocyte of the rat bone marrow. I. The development of the demarcation membrane system and the platelet surface coat. J. Ultrastruct. Res. 24 ~1968! 412–433. @The origin of the platelet membrane in the megakaryocyte.# Breton-Gorius, J. and Guichard, J., Ultrastructural localization of peroxydase activity in human platelets and megakaryocytes. Am. J. Pathol. 66 ~1972! 277–312. @This activity was found to be the earliest indication of megakaryocyte maturation.# Cardiac and Skeletal Muscle The electron microscope played a major role here, revealing a new contraction mechanism, which came to be known as the “sliding filament hypothesis,” associated in particular with the names of Hugh Huxley, Andrew Huxley, and Jean Hanson. A very early investigation of myosin with von Ardenne’s microscope is first recorded. Ardenne, M. von and Weber, H.H., Elektronenmikroskopische Untersuchung des Muskeleiweißkörpers “Myosin”. Kolloid Z. 97 ~1941! 322–325. Bennet, H.S. and Porter, K.R., An electron microscope study of sectioned breast muscle of the domestic fowl. Am. J. Anat. 93 ~1953! 61–105. Hanson, J. and Huxley, H.E., The structural basis of contraction in striated muscles. Symp. Soc. Exp. Biol. 9 ~1955! 228–264. @Demonstration of the sliding-filament mechanism.# Huxley, H.E., The double array of filaments in crossstriated muscle. J. Biophys. Biochem. Cytol. 3 ~1957! 631–648. Huxley, A.F. and Taylor, R.E., Local activation of striated muscle fibres. J. Physiol. 144 ~1958! 426–441. Kidney: Renal Tubules, Glomerules, Juxtaglomerular Apparatus Some of the very early descriptions were given by Oberling’s group in Villejuif and by D.C. Pease and R.F. Baker in the United States. They corresponded to the first images of the complex imbrication between the glomerular basement membrane and the pseudopods of adjacent cells. Gautier, A., Bernhard, W. and Oberling, C., Sur l’existence d’un appareil lacunaire pericapillaire du glomerule de Malpighi révèlé par le microscope électronique. C. R. Soc. Biol. 144 ~1950! 1605–1607. Pease, D.C. and Baker, R.F., Electron microscopy of the kidney. Am. J. Anat. 87 ~1950! 349–389. Among the many other pioneers in this domain were H. Latta, A.B. Maunsbach, J. Rhodin, and J.L. Ericsson; an abundant bibliography is to be found in Ultrastructure of the Kidney ~Dalton, A.J. and Haguenau, F., eds; Academic Press, New York 1967!. In 1959, the first descriptions in the electron microscope of a specific system that regulates arterial pressure in animals and man was a major finding. The structure discovered was termed the neuromyoarterial juxtaglomerular ap- 128 F. Haguenau et al. paratus of the kidney by N. Goormaghtigh in 1937 and had been first described by Camillo Golgi in 1889. Bohle, A., Elektronenmikroskopische Untersuchungen über die Struktur des Gefässpols der Niere. Verh. Deutsch. Ges. Pathol. 43 ~1959! 219–225. Oberling, C. and Hatt, P.-Y., Étude de l’appareil juxtaglomerulaire du rat au microscope électronique. Ann. Anat. Pathol. 5 ~1960! 441–474. Latta, H. and Maunsbach, A.B., The juxtaglomerular apparatus as studied electron microscopically. J. Ultrastruct. Res. 6 ~1962! 547–561. Osborne, M.J., Droz, B., Meyer, P. and Morel, F., Angiotensin II, renal localization in glomerular mesangial cells by autoradiography. Kidney Int. 8 ~1975! 245–254. @This was the first demonstration of the sites of angiotensin receptors.# Secretory Cell, Lysosomes Rinehart, J.F. and Farquhar, M.G., Electron microscopic studies of the anterior pituitary gland. J. Histochem. Cytochem. 1 ~1953! 93–113. @Description of the organization of the endoplasmic reticulum in a glandular cell.# Novikoff, A.R., Beaufay, H. and de Duve, C., Electron microscopy of lysosome-rich fractions from rat liver. J. Biophys. Biochem. Cytol. 2 ~1956! 179–184. @Ultrastructural studies of the phosphatase-rich particle isolated by C. de Duve and his coworkers in 1955.# Caro, L.G. and Palade, G.E., Protein synthesis, storage and discharge in the pancreatic exocrine cell: an autoradiographic study. J. Cell Biol. 50 ~1964! 561–570. Novikoff, A.B. and Novikoff, P.M., Cytochemical contribution to differentiating GERL from the Golgi apparatus. Histochem. J. 9 ~1977! 525–551. Tougard, C., Picart, R. and Tixier-Vidal, A., Electronmicroscopic cytochemical studies on the secretory process in rat prolactin cells in primary culture. Am. J. Anat. 158 ~1980! 471–490. Orci, L., Amherdt, M., Madsen, A., Vassali, G. and Perrelet, A., Direct identification of prohormone conversion site in insulin secreting cell. Cell 42 ~1985! 671–681. Griffiths, G. and Simon, K., The trans-Golgi network sorting at the exit side of the Golgi complex. Science 234 ~1986! 438–446. @Evolution of the GERL concept.# Cerebral Cortex, Nerve Cells (Myelin Sheath), Interneural and Intermuscular Synapses Besides the elucidation of the structure of the neurones ~Nissl bodies! or that of the peripheral nerve sheaths, the electron microscope played a major role in the domain of neuromuscular junctions. Both the American and the French scientists finally put an end to the old debate between the supporters of the continuity or the contiguity between the nerve endings and the skeletal muscle fibers. Geren, B.B., The formation from the Schwann cell surface of myelin in the peripheral nerves of chick embryos. Exp. Cell Res. 7 ~1954! 558–662. Palay, S.L. and Palade, G.E., The fine structure of neurons. J. Biophys. Biochem. Cytol. 1 ~1955! 69–88. Luse, S.A., Electron microscopic observations of the central nervous system. J. Biophys. Biochem. Cytol. 2 ~1956! 531–541. Robertson, J.D., The ultrastructure of the adult vertebrate peripheral myelinated nerve in relation to myelogenesis. J. Biophys. Biochem. Cytol. 1 ~1955! 271–278. de Robertis, E.D.P and Bennett, H.S., Some features of the submicroscopic morphology of synapses in frog and earthworm. J. Biophys. Biochem. Cytol. 1 ~1955! 47–58. Palay, S.L., Synapses in the central nervous system. J. Biophys. Biochem. Cytol. 2 ~1956! 193–202. Couteaux, R., Observations sur l’ultrastructure de la jonction musculo-tendineuse. C.R. Acad. Sci. Paris 249 ~1959! 964–966. Beaudet, A. and Descarries, L., The monoamine innervation of rat cerebral cortex, synaptic and non-synaptic axon terminals. Neuroscience 3 ~1978! 851–860. @Discovery of the morphology of nonsynaptic axon terminals in the brain, a very surprising observation.# 4.4. Ultrastructure of Viruses Among the innumerable topics in the life sciences that have been studied with the electron microscope, viruses History of Electron Microscopy have been selected here because they are a perfect example of a type of specimen that could be visualized in no other way. They thus illustrate in a particularly striking manner the progress accomplished in the realm of biology with this instrument. The first microscopes ~Siemens! were used to study viruses among many other types of specimens. For a full list of references from the various German groups, see the article by Wolpers ~cited in Section 2!, who writes: “At the congress on cell research in 1938 in Zürich, Helmut Ruska reported comprehensively on the first objects studied: bacterial flagellates, bacterial nucleosides, the square form of the myxoma virus in rabbits, the rod-shaped tobacco mosaic virus, the chloroplast structure and the variation of shape in thrombocytes with the structure of fibrin during coagulation.” Some of the references cited by Wolpers are included below. One Siemens microscope went to Rome for a few months and numerous results obtained with it were published, in Italian journals especially. For a list of these, see the essay by Donelli et al. in Section 4.6. The present section is organized by type of virus, but we first list a few of those early papers. 1938 The particle nature of viruses, their size and shape are shown for the first time using the ectromeliavirus, a member of the poxvirus family. Borries, B. von, Ruska, E. and Ruska, H., Bakterien und Virus in übermikroskopischer Aufnahme. Klin. Wochenschr. 17 ~1938! 921–925. 1939 Helmut Ruska summarizes the first results obtained with the new Siemens microscope and discusses its usefulness in virology. Ruska, H., Übermikroskopische Darstellung organischer Struktur ~vom Größenbereich der Zelle bis zum Ultravirus!. Arch. Exp. Zellforsch. 22 ~1939! 673–680. Ruska, H., Borries, B. von and Ruska, E., Die Bedeutung der Übermikroskopie für die Virusforschung. Arch. Ges. Virusforsch. 1 ~1940! 155–169. 1939 Visualization of tobacco mosaic virus, a helically assembled plant virus. Kausche, G.A., Pfankuch, E. and Ruska, H., Die Sichtbarmachung von pflanzlichem Virus im Übermikroskop. Naturwissenschaften 27 ~1939! 292–299. 1940 Visualization of the lytic interaction of phages with the host bacterium. 129 Ruska, H., Die Sichtbarmachung der bakteriophagen Lyse im Übermikroskop and Über ein neues bei der bakteriophagen Lyse auftretendes Formelement. Naturwissenschaften 28 ~1940! 45–46 and 29 ~1940! 367– 368. 1941 An immunological technique is employed for the electron microscope analysis of viruses. Anderson, T.F. and Stanley, W.M., A study by means of the electron microscope of the reaction between tobacco mosaic virus and its antiserum. J. Biol. Chem. 139 ~1941! 339–344. 1943 The Istituto Superiore di Sanità in Rome had acquired one of the early Siemens microscopes but this is confiscated and returned to Germany in 1943. After this, members of the Enrico Fermi School of Physics build a home-made instrument, which continues to function for several years after the war. Babudieri, B. and Bocciarelli, D., Ricerche di microscopia elettronica. I. Studio morfologico di Rickettsia prowazeki. II. Studio morfologico del genere Spironema. Rend. Ist. Super. Sanità 6 ~1943! 298–304 and 305–314. Various types of virus are now presented, classified according to the recommendations of the Seventh Report of the International Committee on Taxonomy of Viruses ~M.H.V. van Regenmortel et al., Virus Taxonomy, Academic Press, San Diego 2000!. The selection here represents the specialization and interest of the compiler and has been made on the basis of their oncogenic potential—a complete list would have been unacceptably long. The literature contains many other excellent books and reviews on classification. DNA Viruses Adenoviruses, adenoviridae Soon after the early work on thin sections, the remarkable icosahedral form of these naked or enveloped viruses was revealed by the use of the negative contrast stains developed by S. Brenner and R.W. Horne in 1959. Morgan, C., Howe, C., Rose, H.M. and Moore, D.H., Structure and development of viruses observed in the electron microscope. IV. Viruses of the RI–APC group @later termed “adenoviruses”#. J. Biophys. Biochem. Cytol. 2 ~1956! 351–360. 130 F. Haguenau et al. Valentine, R.C. and Hopper, P.K., Polyhedral shape of adenovirus particles as shown by electron microscopy. Nature 180 ~1957! 928. Horne, R.W., Brenner, S., Waterson, A.P. and Wildy, P., The icosahedral form of an adenovirus. J. Mol. Biol. 1 ~1959! 84–86. Valentine, R.C. and Pereira, H.G., Antigens and structure of the adenoviruses. J. Mol. Biol. 13 ~1965! 13–20. The so-called papova viruses (papilloma, polyoma, and SV-40 vacuolating virus) Some members of this family have oncogenic properties. Banfield et al., Stone et al., and Kahler et al. were the first to demonstrate the presence of the virion in infected tissues. Banfield, W.G., Dawe, C.J. and Brindley, D.C., Intracellular and extracellular particles in tissue cultures inoculated with parotid-tumor agent ~polyoma virus!. J. Nat. Cancer Inst. 23 ~1959! 1123–1136. Stone, R.S., Shope, R.E. and Moore, D.H., Electron microscope study of the development of the papilloma virus in the skin of the rabbit. J. Exp. Med. 110 ~1959! 543–546. Kahler, H., Rowe, W.P., Lloyd, B.J. and Hartley, J.M., Electron microscopy of mouse parotid tumor ~polyoma!. J. Nat. Cancer Inst. 22 ~1959! 647–657. Croissant, O., Dauguet, C., Jeanteur, P. and Orth, G., Application de la technique d’hybridation in situ à la mise en évidence au microscope électronique de la replication végétative de l’ADN viral dans les papillomes provoqués par le virus de Shope chez le lapin cottontail. C.R. Acad. Sci. Paris 274 ~1972! 614–617. @First example of the use of a molecular biology technique with the electron microscope.# Germond, J.E., Hirt, B., Oudet, P., Gross–Bellard, M. and Chambon, P., Folding of the DNA double helix in chromatin-like structures from SV40. Proc. Natl. Acad. Sci. USA 72 ~1975! 1843–1847. Harris, W.W., Anderson, N.G., Bartlett, T.W., Rutenberg, E.L., McCauley, L.L. and Kinseley, R.M., Unusual particles in human plasma from leukemia and lymphosarcoma. Natl. Cancer Inst. Monographs 21 ~1966! 389–394. No association with type B hepatitis virus was made at the time; the following article represents the first correct identification of the Dane particle and its relation to the Australian antigen, which corresponds to the outer coat protein of the virion; this allowed epidemiological studies to be made and revealed the existence of strong links between chronic infection and liver cancer. Dane, D.S., Cameron, C.H. and Briggs, M., Virus-like particles in serum of patients with Australia-antigenassociated hepatitis. Lancet ~1970! Pt. 1, 695–698. Since Hepadna viruses did not replicate in vitro, knowledge has been acquired essentially through the techniques of molecular biology. Superb images of the hepatitis B genome were thus recorded. Hruska, J.F., Clayton, B.A., Rutenstein, J.L.R. and Robinson, W.S., Structure of hepatitis B Dane particle DNA before and after the Dane particle polymerase reaction. J. Virol. 21 ~1977! 666–672. Charley, P., Pourcel, L., Fritsch, A. and Tiollais, P., Cloning of Escherichia coli and physical structure of B virion DNA. Proc. Natl. Acad. Sci. USA 76 ~1979! 2222–2226. In the domain of liver cancer ~hepatoma!, the following work was of prime importance since it demonstrated the integration of viral DNA in cancer cells. The studies of the group around P. Tiollais, a leader in this field, led to the development of a vaccine. Dejean, A., Brechot, C., Tiollais, P. and Wain-Hobson, S., Characterization of integrated hepatitis B viral DNA cloned from a human hepatoma and hepatoma-derived cell line PLC/PRF/5. Proc. Natl. Acad. Sci. USA 80 ~1983! 2505–2509. Hepatitis viruses (Hepadna viridae) Herpes viruses (herpes viridae) The first electron micrographs of what later came to be called “Dane particle,” which corresponds to the infectious virus, were published by N.G. Anderson and colleagues in 1966. These large ~50–200 nm! double-stranded DNA viruses possess an icosahedral capsid. The core is a toroidal structure. The envelope derives from the inner lamella of the History of Electron Microscopy nuclear envelope, a unique feature. The examples listed here include both the common herpes viruses and others that have oncogenic potential or illustrate some of the first sophisticated studies with molecular biology techniques. Morgan, C., Ellison, S.A., Rose, H.M. and Moore, D.H., Structure and development of viruses observed in the electron microscope. J. Exp. Med. 100 ~1954! 195–201. @Contains early micrographs of HSV-1 in thin sections.# Fawcett, D.W., Electron microscope observations on intracellular virus-like particles associated with the cells of the Lucké renal adenocarcinoma. J. Biophys. Biochem. Cytol. 2 ~1956! 725–742. @A strikingly illustrated article showing the oldest known tumor associated with a herpes virus.# Darlington, R.W. and Moes, L.H., Herpes virus envelopment. J. Virol. 2 ~1968! 48–55. @The envelope of the virus is acquired from newly synthesized regions of the inner lamellae of the nuclear membrane.# Furlong, D., Swift, H. and Zhan, R., Arrangement of the herpes virus deoxyribonucleic acid in the core. J. Virol. 10 ~1972! 1071–1074. @The core is a toroidal structure.# The same remarkable toroidal structure of the core is also illustrated in the cytomegalovirus. Haguenau, F. and Michelson Fiske, S., Cytomegalovirus nucleocapsid assembly and core structure. Intervirology 5 ~1975! 293–299. @This virus is implicated in Karposi’s sarcoma, a tumor commonly seen in AIDS.# The oncogenic potentiality of other Herpes viruses has been shown in many species ~frog, chicken, rabbit, monkey!. In man, herpes viruses were found in tissue cultures derived from Burkitt’s lymphoma ~BL! or in nasopharyngeal carcinoma ~NPC!. Epstein, M.A., Achong, B.J. and Barr, Y.M., Virus particles in cultured lymphoblasts from Burkitt lymphoma. Lancet, i ~1964! 702–703. De Thé, G., Ambrosioni, J.C., Ho, H.C. and Kwan, H.C., Lymphoblastic transformation and presence of Herpes type viral particles in Chinese nasopharyngeal cultures in vitro. Nature 221 ~1969! 770–771. An early example of the use of electron microscopy in molecular biology is illustrated by the study of viral genome 131 structures and the arrangement of nucleotide sequences in the DNA of herpes simplex virus ~HSV-1!, a model widely confirmed. Sheldrick, P. and Berthelot, N., Inverted repetitions in the chromosome of Herpes simplex virus. Cold Spring Harbor Symp. Quant. Biol. 39 ~1974! 667–678. Oncogenic RNA Viruses, Retroviruses One of the most spectacular contributions of electron microscopy to the study of viruses was the morphological demonstration that a characteristic type of RNA virus is involved in avian and murine sarcomas and leukaemias. The viruses of this new family were termed Retroviruses because they contained a newly described enzyme, reverse transcriptase, for which discovery Howard Temin was awarded the Nobel Prize in 1975. The first studies were those of the Rockefeller group, in collaboration with Albert Claude, D.H. Moore, and C. Oberling’s group, in which W. Bernhard headed the electron microscope department at the Institut de Recherches sur le Cancer in Villejuif, France. Fundamental work in the field was achieved in parallel by the group of Joseph W. Beard who, with his wife Dorothy, headed a reputed team and created an important poultry research center in Durham, NC. Avian oncogenic viruses Claude, A., Porter, K.R. and Pickels, E.G., Electron microscope study of chicken tumor cells. Cancer Res. 7 ~1947! 421–430. @Contains some of the earliest micrographs of an RNA sarcoma virus ~RSV!.# Sharp, D.G., Enumeration of virus particles by electron microscopy. Proc. Soc. Exp. Biol. Med. 70 ~1949! 54–59. @First enumeration of virus particles in the electron microscope by comparison with standard particles of known concentration. The number of particles was shown to be related to infectivity.# Bernhard, W., Dontcheff, A., Oberling, C. and Vigier, P., Corpuscules d’aspect virusal dans les cellules de sarcoma de Rous. Bull. Cancer 40 ~1953! 311–321. Gaylord, W.H., Virus-like particles associated with the Rous sarcoma as seen in sections of the tumour. Cancer Res. 15 ~1955! 80–83. @Early descriptions from laboratories other than the Rockefeller Institute and Villejuif.# 132 F. Haguenau et al. Murine oncogenic viruses The existence of a “milk factor” was well established but its identification as a viral particle remained to be shown. The leaders in electron microscopy were D.H. Moore and coworkers at the Rockefeller Institute. Porter, K.R. and Thompson, H.P., A particulate body associated with epithelial cells cultured from mammary carcinoma of mice of a milk factor strain. J. Exp. Med. 88 ~1948! 15–24. Bernhard, W., Bauer, A., Guérin, M. and Oberling, C., Étude au microscope électronique de corpuscules d’aspect virusal dans les epitheliomes mammaires de la souris. Bull. Cancer 42 ~1955! 163–178. Moore, H., Lasfargues, E.Y., Murray, M.R., Haagensen, C.D. and Pollard, E.C., Correlation of physical and biological properties of mouse mammary tumour agent. J. Biophys. Biochem. Cytol. 5 ~1959! 85–92. The “budding process” in leukemia is particularly well illustrated by H. de Harven and C. Friend. De Harven, H. and Friend, C., Further electron microscope studies of a mouse leukaemia induced by cellfree filtrates. J. Biophys. Biochem. Cytol. 7 ~1960! 747– 752. Human retroviruses An outstanding contribution of electron microscopy in the domain of human retroviruses is represented by the pioneering work accomplished in Japan, the United States, and France. Retroviruses were found to be implicated in some types of human leukaemia and it was also shown, a remarkable advance, that they were involved in a newly discovered morbid entity, the acquired immuno-deficiency syndrome ~AIDS!. Poiesz, B.J., Ruscetti, F.W., Reitz, M.S., Kalyahazaman, V.S. and Gallo, R.C., Isolation of a new type C retrovirus ~HTLV! in primary cultured cells of a patient with Sezary’s T-cell leukemia. Nature 294 ~1981! 268– 271. Miyoshi, I., Kubonishi, I., Yoshimoto, S., Akagi, T., Ohtsuki, Y., Shiraishi, Y., Nagata, K. and Hinuma, Y., Type C virus particles in a cord T-cell line derived by co-cultivating normal human cord leucocytes and human leukaemic T cells. Nature 294 ~1981! 770–771. Barré–Sinoussi, F., Chermann, J.C., Rey, F., Nugeyre, M.T., Chamaret, S., Ginest, J., Dauguet, C., Axler-Blin, C., Vezinet–Brun, E., Rouzioux, C., Rozenbaum, W. and Montagnier, L., Isolation of a T lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome ~AIDS!. Science 220 ~1983! 868–871. This first publication was followed by many others and has had a tremendous impact. The new retrovirus was shown to possess ultrastructural features that differ from the standard retroviruses; it belongs to the lentivirus family. 4.5. Plants Plant cell studies have benefited from the era of electron microscopy just like any other eukaryotic cells. Early studies were mainly concerned with the visualization of the fibrillar architecture of the cell walls and the structure of chloroplasts ~visualization of grana and membrane stacks! and with establishing the main characteristics of plant material. 1948–1952 Early studies on plant cell walls. Frey-Wyssling, A. and Muhlethaler, K., Mikrofibrillenbau der pflanzlichen Zellwande. Experientia 4 ~1948! 475. Muhlethaler, K., Electron micrographs of plant fibres. Biochim. Biophys. Acta 3 ~1949! 15–25. Hodge, A.J. and Wardrop, A.B., An electron microscope investigation of the cell wall organization of conifer tracheids and conifer cambium. Austral. J. Sci. Res. B 3 ~1950! 265–269. Liese, W., Demonstration elektronenmikroskopischer Aufnahmn von Nadelhozüpfeln. Ber. Deutsch. Bot. Ges. 64 ~1951! 31–32. @Walter Liese continued to study the vascularization of bamboos.# Harada, H. and Miyazaki, Y., The electron microscopic observation of the cell wall of conifer tracheids. J. Japan. Forest. Soc. 11 ~1952! 350–352. Frey-Wyssling, A., Muhlethaler, K. and Bosshard, H.H., Das Elektronenmikroskop im Dienste der Bestimmung von Pinusarten. Holz Roh-Werkstoff 13 ~1952! 245–249. 1952 Early studies on plant cilia. Manton, I. ~1952!. The fine structure of plant cilia. Symp. Soc. Exp. Biol. 6, 306–319. History of Electron Microscopy 1953 Description of chloroplasts ~1949! and of cell walls. Frey-Wyssling, A., Submicroscopic Morphology of Protoplasm ~Elsevier, Amsterdam 1953!. 1957 Study of the Golgi apparatus in root-tip cells. Buvat, R., Formations de Golgi dans les cellules radiculaires d’Allium cepa; Relations entre l’ergastoplasme et l’appareil vacuolaire. C. R. Acad. Sci. Paris 244 ~1957! 1401–1403 and 245 ~1957! 350–352. 1957–1958 Replica studies of leaf surfaces. 133 1965 In situ fixation of plant cells with aldehydes. Buvat, R., Les infrastructures et la differenciation des cellules criblées de Curcubita pepo. Port. Acta Biol. 7 ~1965! 249–299. 1966 Negative staining of plant cells. Cunningham, W.P., Morré, D.J. and Mollenhauer, H.H., Structure of isolated plant Golgi apparatus revealed by negative staining. J. Cell Biol. 28 ~1966! 169–179. 1966–1967 Classic observations of plant material. Bradley, D.E. and Juniper, B.E., Electron microscopy of leaf surfaces. Nature 180, ~1957! 330–331; Juniper, B.E. and Bradley, D.E., The carbon replica technique in the study of the ultrastructure of leaf surfaces. J. Ultrastruct. Res. 2 ~1958! 16–27. Manton, I., Observations on scale formation in Prymnesium parvum. J. Cell Sci. 1 ~1966! 375–380; Manton, I., Further observations on scale formation in Chrysochromulina chiton. J. Cell Sci. 2 ~1967! 411–418. 1960–1961 Seminal studies on the Golgi apparatus in plant cells. 1968 First application of the polysaccharide labeling technique, introduced by Thiéry, to the study of plant cell walls. Manton, I., On a reticular derivative from Golgi bodies in the meristem of Anthoceros. J. Biophys. Biochem. Cytol. 8 ~1960! 221–231. Mollenhauer, H.H., Whaley, W.G. and Leech, J.H., A function of the Golgi apparatus in the onion root tip cells. J. Ultrastruct. Res. 5 ~1961! 193–200. 1961 An early examination of phloem. Esau, K. and Cheadle, V.I., An evaluation of studies on ultrastructure of sieve plates. Proc. Natl. Acad. Sci. USA 47 ~1961! 1716–1736. 1963 The first description of microtubules in plant cells. Ledbetter, M.C. and Porter K.R., A “microtubule” in plant cell fine structure. J. Cell Biol. 19 ~1963! 239– 250. 1964–1968 Early cryomicroscopy of plant material. Branton, D. and Moor, H., Fine structure in freezeetched Allium cepa L. roots. J. Ultrastruct. Res. 11 ~1964! 401–411. Roland, J.C. and Sandoz, D., Détection cytochimique des sites de formation de polysaccharides prémembranaires dans les cellules végétales. J. Microsc. 8 ~1968! 263–268. 1969 Scanning microscopy of plant replicas. Idle, D.B., Scanning electron microscopy of leaf surface replicas and the measurement of stomatal aperture. Ann. Bot. 33 ~1969! 75–76. 1971–1973 Developments in cryomethods for plant studies. Robards, A.W. and Parish, G.R., Preparation and mechanical requirements for freeze etching thick walled plant tissue. J. Microscopy 93 ~1971! 61–66. Buttrose, M.S., Ultrastructure of barley aleurone cells as shown by freeze-etching. Planta 96 ~1971! 13–26. Fineran, B.A., Fracture faces of tonoplast in root tips after various conditions of pretreatment prior to freeze etching. J. Microscopy 96 ~1972! 333–342. Hall, D.M., Wax microchannels in the epidermis of white clover. Science 158 ~1967! 505–506. Hereward, F.V. and Northcote, D.H., Fracture planes of plasmalemma of some higher plants revealed by freeze-etch. J. Cell Sci. 13 ~1973! 621–635. Northcote, D.H. and Lewis, D.R., Freeze-etched surfaces of membranes and organelles in the cells of pea root tips. J. Cell Sci. 3 ~1968! 199–206. Johnson, R.P.C. Filaments but not membranous transcellular strands in sieve pores in freeze-etched translocating phloem. Nature 244 ~1973! 464–466. 134 F. Haguenau et al. Cox, G. and Juniper, B., Electron microscopy of cellulose in entire tissue. J. Microscopy 97 ~1973! 343–355. Robards, A.W., Freeze-etching of plant cells. Mikroskopie 34 ~1978! 12–18. Barlow, K.K., Buttrose, M.S., Simmonds, D.H. and Vesk, M., The nature of the starch protein interface in wheat endosperm. Cereal Chem. 50 ~1973! 443, 454. Willison, J.H.M. and Brown, R.M., Pretreatment artefacts in plant cells. In Freeze-fracture: Methods, Artefacts and Interpretations ~Rash, J.E. and Hudson, C.S., eds! 51–57 ~Raven Press, New York 1979!. 1972 The three-dimensional structure of wood revealed by the scanning electron microscope. Meylan, B.A. and Butterfield, B.G., Three-dimensional Structure of Wood: A Scanning Electron Microscope Study ~Chapman and Hall, London 1972!. 1974–1983 Use of the high-voltage electron microscope to study plant specimens. Poux, N., Favard, P. and Carasso, N., Étude en microscopie electronique à haute tension de l’appareil vacuolaire dans les cellules méristèmatiques de racines de concombre. J. Microscopie 21 ~1974! 173–180. Harris, N., Endoplasmic reticulum in developing seeds of Vicia faba. A high voltage electron microscopy. Planta 146 ~1979! 63–69. Hawes, C.R., Applications of high voltage electron microscopy to botanical ultrastructure. Micron 12 ~1981! 227–257. Cox G. and Juniper B., High-voltage electron microscopy of whole critical point dried plant cells: Fine, cytoskeletal elements in the moss Bryum tenuisitum. Protoplasma 115 ~1983! 70–80. Marty, F., Microscopie electronique à haute tension de l’appareil provacuolaire dans les cellules méristèmatiques des racines de radis Raphinus sativus L. Ann. Sci. Nat. Bot. (Paris) 13, ~1983! 245–260. 1974 Preparation methods for wood. Exley, R.R., Butterfield B.G. and Meylan B.A., Preparation of wood specimens for the scanning electron microscope. J. Microscopy 101 ~1974! 21–30. 1978 A good survey of SEM techniques for plant cell studies. Robards, A.W., An introduction to techniques for scanning electron microscopy of plant cells. In Electron Microscopy and Cytochemistry of Plant Cells ~Hall, J.L., ed.! 343–415 ~Elsevier North Holland Biomedical Press, Amsterdam 1978!. 1978–1992 Steady progress in the development of cryotechniques for plant studies. Wilson, A.J. and Robards, A.W., Some experiences in the use of polymeric cryoprotectant in the freezing of plant tissue. J. Microscopy 125 ~1981! 287–298. Harvey, D.M.R., Freeze-substitution. J. Microscopy 127 ~1982! 209–222. Platt-Aloia, K.A. and Thomson, W.W., Freeze-fracture of intact plant tissues. Stain Technol. 57 ~1982! 327– 334. Mueller, S.C. and Brown, R.M., The control of cellulose microfibril deposition in the cell wall of higher plants: II. Freeze-fracture microfibril patterns in maize seedling tissues following experimental alteration with colchicine and ethylene. Planta 154 ~1982! 501–515. Robards, A.W. and Clarkson, D.T., Effects of chilling temperatures on root cell membranes as viewed by freeze fracture electron microscopy. Protoplasma 122 ~1984! 75–85. McCully, M.E. and Canny, M.J., The stabilization of labile configurations of plant cytoplasm by freezesubstitution. J. Microscopy 139 ~1985! 27–33. Gilkey, J.C. and Staehelin, L.A., Ultrarapid freezing for the preservation of cellular ultrastructure. J. Electron Microsc. Techn. 3 ~1986! 177–210. Lancelle, S.A., Callaham, D.A. and Hepler, P.K., A method for the rapid freeze fixation of plant cells. Protoplasma 131 ~1986! 153–165. Hawes, C. and Martin, B., Deep-etching of plant cells: cytoskeleton and coated pits. Cell Biol. Int. Repts. 10 ~1986! 985–992. Hirsh, A.G., Vitrification in plants as a natural form of cryoprotection. Cryobiology 24 ~1987! 214–228. Jeffree, C.E., Read, N.D., Smith, J.A.C. and Dale, J.E., Water droplets and ice deposits in leaf intercellular spaces: Redistribution of water during cryofixation for scanning electron microscopy. Planta 172 ~1987! 20–37. History of Electron Microscopy Sargent, J.A., Low temperature scanning electron microscopy—advantages and applications. Scanning Microsc. 2 ~1988! 835–849. Weibull, C. and Albertsson, P.A., Ultrastructure of spinach thylakoids as seen in low temperature and conventional embeddings. J. Ultrastruct. Molec. Struct. Res. 100 ~1988! 55–59. Studer, D., Michel, M. and Müller, M., Cryofixation of plant tissues by high pressure freezing. EUREM-9 ~York, 1988!. Institute of Physics Conference Series 93, vol. 3 ~1988! 19–20. Craig, S. and Staehelin, L.A., High pressure freezing of intact plant tissues. Evaluation and characterisation of novel features of the endoplasmic reticulum and associated membrane systems. Eur. J. Cell Biol. 46 ~1988! 80–93. Kaeser, W., Freeze substitution of plant tissues with a new medium containing dimethoxypropane. J. Microscopy 154 ~1989! 273–278. Kaeser, W., Koyro, H.W. and Moor, H., Cryofixation of plant tissues without pretreatment. J. Microscopy 154 ~1989! 279–288. McCann, M.C., Wells, B. and Roberts, K., Direct visualisation of cross-links in the primary plant cell wall. J. Cell Sci. 96 ~1990! 323–334. Lichtscheid, I.K., Lancelle, S.A., and Hepler, P.K., Actinendoplasmic reticulum complexes in Drosera. Their structural relationship with the plasmalemma, nucleus and organelles in cells prepared by high pressure freezing. Protoplasma 155 ~1990! 116–126. Satiat-Jeunemaître, B., Martin, B. and Hawes, C., Plant cell wall architecture is revealed by rapid-freezing and deep-etching. Protoplasma 167 ~1992! 33–42. 1979 Three-dimensional studies on the plant cell wall. Roland, J.C. and Vian, B., The wall of growing plant cells: its three-dimensional organization. Int. Rev. Cytol. 61 ~1979! 129–166. 135 1980 The mitotic apparatus of plant cells. Hepler, P.K., Membranes of the mitotic apparatus of barley cells. J. Cell Biol. 86 ~1980! 490–499. 1981–1982 Thiéry’s technique is combined with the controlled extraction of polysaccharides to reveal the highly organized network of the primary cell walls during growth. Reis, D., Cytochimie ultrastructurale des parois en croissance par extraction ménagée. Effets comparés du diméthylsulfoxyde et de la methylamine sur le démasquage des textures. Ann. Sci. Nat. Bot. (Paris) 2–3 ~1981–2! 121–136. 1982 A valuable assessment of staining techniques for plants. Schnepf E., Hausmann K. and Herth W., The osmium tetroxide-potassium ferrocyanide OsFeCN staining techniques for electron microscopy: A critical evaluation using ciliates, algae, mosses and higher plants. Histochemistry 76 ~1982! 261–271. 1982 Dictyosomes and the endoplasmic reticulum. Juniper, B.E., Hawes, C.R. and Horne, J.C., The relationship between the dictyosomes and the forms of the endoplasmic reticulum in plant cells with different export programs. Bot. Gaz. 143 ~1982! 135–145. 1983 Use of the ZIO technique for plants. Harris, N. and Oparka, K., Connections between dictyosomes, ER and GERL in cotyledons of mung bean Vigna radiata L. Protoplasma 114 ~1983! 93–102. 1983 Preparation of plant surfaces for SEM. Sargent, J.A., The preparation of leaf surfaces for scanning electron microscopy: A comparative study. J. Microscopy 129 ~1983! 103–110. 1983–1984 The first two examples in plant studies of the use of enzyme–gold markers following the technique described by Roth and Bendayan. 1980 A preparation technique for ion localization in botanical specimens. Vian, B., Brillouet, J.M., and Satiat-Jeunemaître, B., Ultrastructural visualisation of xylans in cell walls of hardwood by means of xylanase gold complex. Biol. Cell 49, ~1983! 179–182. Harvey, D.M.R., The preparation of botanical samples for ion localisation studies at subcellular level. Scanning Electron Microsc. ~1980! Part 2, 409–420. Ruel, K. and Joseleau, J.P., Use of enzyme-gold complexes for the structural localisation of hemicelluloses in the plant cell wall. Histochemie 81 ~1984! 573–580. 136 F. Haguenau et al. 1984 Critical-point drying/dry cleaving in plant cells. Traas, J.A., Visualisation of the membrane bound cytoskeleton and coated pits of plant cells by means of dry cleaving. Protoplasma 119 ~1984! 212–218. 1984–1992 Examples of immunogold staining in plants. Greenwood, J.S., Keller, G.A. and Chrispeels, M.J., Localization of phytohemagglutinin in the embryonic axis of Phaseolus vulgaris with ultrathin cryosections embedded in plastic after indirect immunolabeling. Planta 162 ~1984! 548–555. Craig, S. and Goodchild, D.J., LR White resin and improved on-grid immunogold detection of vicilin, a pea storage protein. Cell Biol. Int. Repts. 8 ~1984! 879–886. Immunogold labelling of plant Golgi apparatus after low-temperature embedding in the acrylic resin LR White; the latter is less toxic than the Lowicryl resin commonly used for animal cells. 1986 Three-dimensional studies on the endoplasmic reticulum in plant cells. Stephenson, J.L.M. and Hawes, C.R., Stereology and stereoscopy of endoplasmic reticulum in the root cap cells of maize. Protoplasma 131 ~1986! 32–46. 1987 Progress in understanding vesicle structure. Coleman J., Evans D., Hawes C., Horsley D. and Cole L., Structure and molecular organization of higher plant coated vesicles. J. Cell Sci. 88 ~1987! 35–45. 1989 A good selection of source papers on lectins is to be found here. Benhamou, N., Preparation and application of lectin– gold complexes. In Colloidal Gold, Principles, Methods and Applications ~Hayat, M.A., ed.!, pp. 95–143 ~Academic Press, New York 1989!. 1991 Affinity probes. Sossountzov, L., Sotta, B., Maldiney, R., Sabbagh, I. and Miginiac, E., Immuno-electron microscopy localization of abcissic acid with colloidal gold on Lowicrylembedded tissues of Chenopodium polyspermum L. Planta 168 ~1986! 471–481. Van den Bosch, K.A., Light and electron microscopic visualisation of uricase by immunogold labelling of sections of resin-embedded soybean nodules. J. Microscopy 143 ~1986! 187–197. Traas, J.A. and Kengen, H.M.P. Gold labelling of microtubules in cleaved whole mounts of cortical root cells. J. Histochem. Cytochem. 34 ~1986! 1501–1504. Lancelle, S.A. and Hepler, P.K., Immunogold labelling of actin on sections of freeze-substituted plant cells. Protoplasma 150 ~1989! 172–174. Satiat-Jeunemaître, B. and Hawes, C., Redistribution of a Golgi glycoprotein in plant cells treated with brefeldin A. J. Cell Sci. 103 ~1992! 1153–1156. 1984 Chloroplast structure elucidated in the SEM. Barnes, S.H. and Blackmore, S., Scanning electron microscopy of chloroplast ultrastructure. Micron Microsc. Acta 15 ~1984! 187–194. 1985 Plant cell fixation. Coetzee, J., Fixation of plant cells for electron microscopy. In Botanical Microscopy 1985 ~Robards, A.W., ed.! 17–38 ~Oxford University Press, Oxford 1985!. Roy, S. and Vian, B., Transmural exocytosis in maize root cap: visualization by simultaneous use of a cellulose probe and a fucose probe. Protoplasma 161 ~1991! 181–191. 1991–1994 Elucidation of scale formation. Melkonian, M., Becker, B. and Becker D., Scale formation in algae. J. Electron Microsc. Tech. 17 ~1991! 165–178. Becker, B., Marin, B. and Melkonian, M., Structure, composition and biogenesis of prasinophyte cell coverings. Protoplasma 181 ~1994! 233–244. Lavan, S. and Wetherbee, R., Structure and development of the scale case of Mallomonas adamas ~Synurophyceae!. Protoplasma 181 ~1994! 259–268. 1991 Progress in SEM. Read, N.D. and Jeffree, C.E., Low temperature scanning electron microscopy in biology. J. Microscopy 161 ~1991! 59–72. 1993 The bible of Arabidopsis for molecular biologists; Arabidopsis is to plants what Drosophila is to animals. Dolan, L., Janmaat, K., Willemsen, V., Linstead, P., Poethig, S., Roberts, K. and Scheres, B., Cellular organisation of the Arabidopsis thaliana root. Development 119 ~1993! 71–84. History of Electron Microscopy 1993 Membrane traffic models for plants. Schnepf, E., Golgi apparatus and slime secretion in plants: the early implications and recent models of membrane traffic. Protoplasma 172 ~1993! 3–11. 1996 A rare example of the use of field-emission SEM to study plant cells. Vesk, P.A., Vesk, M. and Gunning, B.E.S., Field emission scanning electron microscopy of microtubule arrays in higher plant cells. Protoplasma 195 ~1996! 168–182. 1999 Three-dimensional structure of the gap junction. Unger, V., Kumar, N., Gilula, N.B. and Yaeger, B., Three-dimensional structure of a recombinant gap junction membrane channel. Science 283 ~1999! 1176–1179. 4.6. Books and Surveys in the Life Sciences 137 Fauré–Fremiet, E., Cils vibratiles et flagelles. Biol. Rev. 36 ~1961! 464–536. Caspar, D.L.D. and Klug, A., Physical principles in the construction or regular viruses. Cold Spring Harbor Symp. Quant. Biol. 27 ~1962! 1–24. Novikoff, A.B., Lysosomes in the physiology and pathology of cells: contributions of staining methods. In CIBA Foundation Symposium on Lysosomes ~de Reuck, A.V.S. and Cameron, M.P., eds!, pp. 36–73 ~Churchill, London 1963!. @First representation of the morphology of lysosomes, which complemented the discovery of their function by Christian de Duve.# Dallner, G., Studies on the structural and enzymic organization of the membranous elements of liver microsomes. Acta Pathol. Microbiol. Scand. Suppl. 166 ~1963! 1–94. Fawcett, D.W., The structure of the mammalian spermatozoon. Int. Rev. Cytol. 7 ~1958! 195–234. Feldherr, C.M., Gall, J.G., Goldstein, L., Harding, C.V., Loewenstein, W.R. and Mirsky, A.E., The nuclear membrane and nucleocytoplasmic exchange. Protoplasmatologia @Handbuch der Protoplasmaforschung# Vol. 5, Karyoplasma ~Nucleus!, Part 2, 72 pp. ~Springer, Vienna & New York 1964!. Haguenau, F., The ergastoplasm: its history, ultrastructure and biochemistry. Int. Rev. Cytol. 7 ~1958! 425– 483. Fawcett, D.W., An Atlas of Fine Structure. The Cell, its Organelles and Inclusions ~Saunders, Philadelphia & London 1966!. Dalton, A.J., Organization in benign and malignant cells. Lab Invest. 8 ~1959! 510–537. Haguenau, F., Ultrastructure of the cancer cell. In The Biological Basis of Medicine ~Bittar, E.E., ed.! Vol. 5, pp. 433–485 ~Academic Press, London & New York 1969!. @It is to be noted that no real difference is observed between the ultrastructure of normal and malignant cells.# Levaditi, C., Images Electroniques en Microbiologie ~Librairie Malsine, Paris, 1949!. Bernhard, W., The detection and study of tumor viruses with the electron microscope. Cancer Res. 20 ~1960! 712–727. Couteaux, R., Motor end-plate structure. In The Structure and Function of Muscle ~Bourne, G.H., ed.!, pp. 337–380 ~Academic Press, New York 1960! Wildy, P., Russell, W.C. and Horne, R.W., The morphology of herpes viruses. Virology 12 ~1960! 204–222. @A review by the British school.# Horne, R.W. and Wildy, P., Symmetry of virus architecture. Virology 15 ~1961! 348–373. Boyd, J.D., Johnson, F.R. and Lever, J.D., eds., Electron Microscopy in Anatomy ~Arnold, London 1961!. Grimstone, A.V., Fine structure and morphogenesis in Protozoa. Biol. Rev. 36 ~1961! 97–150. Dalton, A.J. and Haguenau, F., eds., Ultrastructure of Animal Viruses and Bacteriophages, an Atlas ~Academic Press, New York & London 1973!. Benedetti, E.L. and Favard, P., eds., Freeze-etching, Techniques and Applications ~Société Française de Microscopie Electronique, Paris 1973!. Bessis, M., Living Blood Cells and Their Ultrastructure ~Springer, Berlin 1973!. Tooze, J., The Molecular Biology of Tumour Viruses ~Cold Spring Harbor Laboratory, Cold Spring Harbor 1973!. 138 F. Haguenau et al. Koehler, J.K., ed., Biological Electron Microscopy, Advanced Techniques. I, II, III. ~Springer, Berlin and New York 1973, 1978, 1986!. Hall, J.L. and Hawes, C.R., Electron Microscopy of Plant Cells ~Academic Press, London 1991!. @Working manual of the major modern techniques used to prepare plant material for TEM and SEM.# Gunning, B.E.S. and Steer, M.W., Plant Cell Biology, an Ultrastructural Approach ~Arnold, London 1975!. @Atlas of micrographs with detailed captions on plant cell ultrastructure.# Read, N.D. and Jeffree, C.E., Low temperature scanning electron microscopy in biology. J. Microscopy 161 ~1991! 59–72. Erasmus, D.A., ed., Electron Probe Microanalysis in Biology ~Chapman & Hall, London 1978!. Echlin, P., Low-temperature Microscopy and Analysis ~Plenum, New York 1992!. Hall, J.L., ed., Electron Microscopy and Cytochemistry of Plant Cells ~Elsevier North Holland Biomedical Press, Amsterdam 1978!. Gupta, B.L., The electron microprobe x-ray microanalysis of frozen-hydrated specimens with new information on fluid-transporting epithelia. In Microbeam Analysis in Biology ~Lechene, C.P. and Warner, R.R., eds.! 375–408 ~Academic Press, New York 1979!. Roland, J.C. and Vian, B., The wall of growing plant cells: its three-dimensional organization. Int. Rev. Cytol. 61 ~1979! 129–166. Brack, C., DNA electron microscopy. Crit. Rev. Biochem. 10 ~1981! 113–169. Gibbons, I.R., Cilia and flagella of eukaryotes. J. Cell Biol. 91 ~1981! 107s–124s. Donelli, G., Morace, A., Ardita, G. and Notargiacomo, S., Bibligrafica analitica delle ricerche ultrastrutturale svolte nell’Istituto Superiore di Sanità: 1943–1982. ISTISAN Report No. 1983/46 ~Istituto Superiore di Sanità, Rome 1983!. Gross, L., Oncogenic Viruses ~Pergamon, Oxford, 3rd ed. 1983!. Robards A.W., Botanical Microscopy ~Oxford University Press, Oxford 1985!. Robards, A.W. and Sleytr, U.B., Low Temperature Methods in Biological Electron Microscopy ~Elsevier, Amsterdam 1985!; Volume 10 of “Practical Methods in Electron Microscopy” ~Glauert, A., ed.!. Nermut, B.V. and Stephen, A.C., eds., Animal Virus Structure ~Elsevier, Amsterdam 1987!. Taugner, R. and Hackenthal, E., The Juxtaglomerular Apparatus, Structure and Function ~Springer, Berlin 1989!. Gunning, B.E.S. and Steer, M.W., Plant Cell Biology, Structure and Function ~Jones and Bartlett, Sudbury, MA 1996!. @Includes 405 images and interpretations of plant cell structure obtained by light and electron microscopy.# A CKNOWLEDGMENTS We are most grateful to the very many scientists who were kind enough to send suggestions, comment on drafts of individual sections, and help in a host of other ways. Françoise Haguenau particularly wishes to acknowledge the help of Jean André ~Paris!, F. Basset ~Paris!, E.L. Benedetti ~Paris!, J. Breton–Gorius ~Paris!, Renée Charret ~Paris!, Philippe Compère ~Liège!, René Couteaux ~Paris!, Odile Croissant ~Paris!, E. de Harven ~Brussels!, E. Delain ~Paris!, R. Dourmashkin ~Paris!, B. Droz ~Lausanne!, Stan Fakan ~Lausanne!, Michel Fardeau ~Paris!, Guy Fournier ~Paris!, H. Frank ~Tübingen!, W.W. Franke ~Heidelberg!, JeanMarie Gasc ~Paris!, Audrey Glauert ~Cambridge!, E. Kellenburger ~Lausanne!, G. Lyon ~Louvain!, M.V. Nermut ~London!, G. Palade ~San Diego!, J. Poirier ~Paris!, F. and E. Puvion ~Villejuif!, A. Ryter ~Lausanne!, J. Schrevel ~Paris!, L. Staeckel ~Strasbourg!, J. Taxi ~Paris!, R. Terry ~San Diego!, Daniel Thomas ~Rennes!, E.M.S. Tomé ~Paris!, and J. Witz ~Strasbourg!. We trust that no one will be offended that some of the suggested references have not been included but we felt that the list, already much longer than originally planned, must be kept within reasonable bounds. In making difficult choices, we had ever present in our minds the lines For what there was, none cared a jot But all were wroth with what was not.