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Cryo-electron microscopy of vitrified specimens

  • Jacques Dubochet (a1), Marc Adrian (a1), Jiin-Ju Chang (a1), Jean-Claude Homo (a1), Jean Lepault (a1), Alasdair W. McDowall (a1) and Patrick Schultz (a1)...


Cryo-electron microscopy of vitrified specimens was just emerging as a practical method when Richard Henderson proposed that we should teach an EMBO course on the new technique. The request seemed to come too early because at that moment the method looked more like a laboratory game than a useful tool. However, during the months which ellapsed before the start of the course, several of the major difficulties associated with electron microscopy of vitrified specimens found surprisingly elegant solutions or simply became non-existent. The course could therefore take place under favourable circumstances in the summer of 1983. It was repeated the following years and cryo-electron microscopy spread rapidly. Since that time, water, which was once the arch enemy of all electronmicroscopists, became what it always was in nature – an integral part of biological matter and a beautiful substance.



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Adrian, M., Dubochet, J., Lepault, J. & McDowall, A. W. (1984). Cryo-electron microscopy of viruses. Nature 308, 3236.
Amos, L. A., Henderson, R. & Unwin, P. N. T. (1982). Three-dimensional structure determination by electron microscopy of two-dimensional crystals. Prog. Biophys. molec. Biol. 39, 183231.
Angell, C. A. (1983). Supercooled water. A. rev. phys. Chem. 34, 593630.
Angell, C. A. & Choi, Y. (1986). Crystallization and vitrification in aqueous systems. J. Microsc. 141, 251261.
Bellare, J. R., Davis, H. T., Scriven, L. E. & Talmon, Y. (1986). An improved controlled-environment vitrification system (CEVS) for cryofixation of hydrated TEM samples. In Proc. XIth Int. Conf. Elec. Microsc., Kyoto, vol. 11 (ed. Imura, T., Maruse, S. and Suzuki, T.), pp. 367368.
Bernal, J. D. & Fowler, R. H. (1933). A theory of water and ionic solution with particular reference to hydrogen and hydroxyl ions. J. chem. Phys. 1, 515548.
Bernhard, W. (1965). Ultramicrotomie à basse température. Année biol. 4, 519.
Berry, R. S., Rice, S. A. & Ross, J. (1980). Physical Chemistry. New York: Wiley.
Born, M. & Wolf, E. (1975). Principles of Optics, 5th ed.Oxford: Pergamon Press.
Box, H. C. (1975). Cryoprotection of irradiated specimens. In Physical Aspects of Electron Microscopy and Microbeam Analysis (ed. Siegel, B. M. and Beaman, D. R.), pp. 279285. New York: Wiley.
Brack, C. (1981). DNA electron microscopy. Crit. Rev. Biochem. 10, 113169.
Brüggeller, P. & Mayer, E. (1980). Complete vitrification in pure liquid water and dilute aqueous solutions. Nature 288, 569571.
Burton, E. F. & Oliver, W. F. (1935). The crystal structure of ice at low temperature.Proc. R. Soc. Lond. A 153, 166172.
Cantor, C. R. & Schimmel, R. P. (1980). Biophysical Chemistry, vol. 1–3. San Francisco: Freeman.
Chang, C.-F., Ohno, T. & Glaeser, R. M. (1985). The fatty acid monolayer technique for preparing frozen hydrated specimens. J. elec. Microsc. Techn. 2, 5965.
Chang, J.-J., McDowall, A. W., Lepault, J., Freeman, R., Walter, C. A. & Dubochet, J. (1983). Freezing, sectioning and observation artefacts of frozen hydrated sections for electron microscopy. J. Microsc. 132, 109123.
Chiu, W. (1982). High resolution electron microscopy of unstained hydrated protein crystals. In Electron Microscopy of Proteins, vol. 2 (ed. Harris, J. R.), pp. 233259. London: Academic Press.
Chiu, W. (1986). Electron microscopy of frozen, hydrated biological specimens. A. Rev. Biophys. Chem. 15, 237257.
Christensen, A. K. (1971). Frozen thin sections of fresh tissue for electron microscopy, with a description of pancreas and liver. J. Cell Biol. 51, 772804.
Clegg, J. S. (1982). Alternative views on the role of water in cell function. In Biophysics of Water (ed. Franks, F. and Mathias, S.), pp. 365383. Chichester: Wiley.
Coslett, V. E. (1978). Radiation damage in the high resolution electron microscopy of biological materials: a review. J. Microsc. 113, 113129.
Crew, A. V. (1973). Considerations of specimen damage for the transmission electron microscope, conventional versus scanning. J. molec. Biol. 80, 315325.
Davidson, D. W. (1973). Clathrate hydrates. In Water: A Comprehensive Treatise, vol. 2, (ed. Franks, F.), pp. 115234. New York: Plenum Press.
Davy, J. D. & Branton, D. (1970). Subliming ice surfaces: freeze-etch electron microscopy. Science 163, 12161218.
Dietrich, I., Formanek, H., Fox, F., Knapek, E. & Weyl, R. (1979). Reduction of radiation damage in an electron microscope with superconducting lens system. Nature 277, 380381.
Dietrich, I., Fox, F., Heide, H. G., Knapek, E. & Weyl, R. (1978). Radiation damage due to knock-on processes on carbon foils cooled to liquid helium temperature. Ultramicroscopy 3, 185189.
Dowell, L. G. & Rinfret, A. P. (1960). Low-temperature forms of ice as studied by X-ray diffraction. Nature 188, 11441148.
Dubochet, J. (1975). Carbon loss during irradiation of T4 bacteriophages and E. coli bacteria in electron microscopes. J. Ultrastruct. Res. 52, 276288.
Dubochet, J., Adrian, M., Lepault, J. & McDowall, A. W. (1985). Cryo-electron microscopy of vitrified biological specimens. Trends in Biochem. Sci. 10, 143146.
Dubochet, J., Adrian, M., Schultz, P. & Oudet, P. (1986). Cryo-electron microscopy of vitrified SV40 minichromosomes. The liquid drop model. EMBO J. 5, 519528.
Dubochet, J., Adrian, M., Teixeira, J., Kadiyali, R. K., Alba, C. M., Macfarlane, D. R. & Angell, C. A. (1984). Glass-forming microemulsions: vitrification of simple liquids and electron microscope probing of droplet packing modes. J. phys. Chem. 88, 67276732.
Dubochet, J., Adrian, M. & Vogel, R. H. (1983 a). Amorphous solid water obtained by vapour condensation or by liquid cooling: a comparison. Cryo-Letters 4, 233240.
Dubochet, J., Chang, J.-J., Freeman, R., Lepault, J. & McDowall, A. W. (1982 a). Frozen aqueous suspensions. Ultramicroscopy 10, 5562.
Dubochet, J., Groom, M. & Müller, , Neuteboom, S. (1982 b). The mounting of macromolecules for electron microscopy. In Advances in Optical and Electron Microscopy, vol. 8 (ed. Cosslett, V. E. and Barer, R.), pp. 107135. London: Academic Press.
Dubochet, J. & Kellenberger, E. (1972). Selective adsorption of particles to the supporting film and its consequences on particle counts in electron microscopy. Microscopica Acta 72, 119130.
Dubochet, J. & Lepault, J. (1984). Cryo-electron microscopy of vitrified water J. Physics 45, C7/8594.
Dubochet, J., Lepault, J., Freeman, R., Berriman, J. A. & Homo, J.-CL. (1982 c). Electron microscopy of frozen water and aqueous solutions. J. Microsc. 128, 219237.
Dubochet, J. & McDowall, A. W. (1981). Vitrification of pure water for electron microscopy. J. Microsc. 124, RP3–4.
Dubochet, J. & McDowall, A. W. (1984 a). Frozen hydrated sections. In Science of Biological Specimen Preparation, pp. 147152. Chicago: SEM Inc., AMF O'Hare.
Dubochet, J. & McDowall, A. W. (1984 b). Cryo-ultramicrotomy: study of ice crystals and freezing damage.In Proc. 8th Eur. Congr. Elec. Microsc., Budapest (ed. Csanady, A., Röhlich, P. and Szabo, D.), vol. 2, pp. 14071410. Budapest: Progr. Committee.
Dubochet, J., McDowall, A. W., Menge, B., Schmid, E. N. & Lickfeld, K. G. (1983 b). Electron microscopy of frozen-hydrated bacteria. J. Bact. 155, 381390.
Eisenberg, D. & Crothers, D. (1979). Physical Chemistry with Applications to the Life Sciences. Menlo Park, CA.: Benjamin/Cumming.
Eisenberg, D. & Kauzmann, W. (1969). The Structure and Properties of Water. Oxford University Press.
Erickson, H. P. & Klug, A. (1971). Measurement and compensation of defocusing and aberrations by Fourrier processing of electron microgrpahs. Phil. Trans. R. Soc. Lond. B 261, 105118.
Escaig, J. (1982 a). New instruments which facilitate rapid freezing at 83 K and 6 K. J. Microsc. 126, 221229.
Escaig, J. (1982 a). Ultra-rapid freezing of cells and cellular material: a review of methods.In Proc. 10th Int. Congr. Elec. Microsc., Hamburg, vol. 3, pp. 169176. Frankfurt: Deutsche Ges. für Elecktronenmikroskopie, e.V.
Eusemann, R., Rose, H. & Dubochet, J. (1982). Electron scattering in ice and organic materials, J. Microsc. 128, 239249.
Fahy, G. M., Macfarlane, D. R., Angell, C. A. & Meryman, H. T. (1984). Vitrification as an approach to cryo-preservation., Cryobiology 21, 407426.
Fernandez-Moran, H. (1960). Low temperature preparation techniques for electron microscopy of biological specimens based on rapid freezing with liquid helium II. Ann. N. Y. Acad. Sci. 85, 689713.
Fernandez–Moran, H. (1966). High-resolution electron microscopy with super-conducting lenses at liquid helium temperatures.Proc. Natn. Acad. Sci. U.S.A. 56, 801808.
Fernandez-Moran, H. (1985). Cryo-electron microscopy and ultramicrotomy: Reminiscences and reflections. In Advances in Electronics and Electron Physics, supplement 16, pp. 167223. New York: Academic Press.
Feynman, R. P., Leighton, R. B. & Sands, M. (1965). Lectures on Physics, vol. I–III. Reading, Massachusetts: Addision-Wesley.
Finney, J. L. (1986). The role of water perturbations in biological process. In Water and Aqueous Solutions (ed. Neilson, G. W. and Enderby, J. E.), pp. 227244. Bristol: Hilger.
Frank, H. S. (1970). The structure of ordinary water. Science 169, 635641.
Franks, F. (19721982). Water: A Comprehensive Treatise, vol. 1–7 (ed. Franks, F.). New York: Plenum Press.
Franks, F. (1982). The properties of aqueous solutions at subzero temperatures. In Water: A Comprehensive Treatise, vol. 7 (ed. Franks, F.), pp. 215338. New York: Plenum Press.
Frederik, P. M., Busing, W. M. & Persson, A. (1982). Concerning the nature of the cryosectioning process. J. Microsc. 125, 167175.
Frederik, P. M., Busing, W. M. & Persson, A. (1984). Surface defects on thin cryosections. In Scanning Electron Microscopy, vol. 1, pp. 433443. Chicago: SEM Inc., AMF O'Hare.
Freeman, R., Leonard, K. R. & Dubochet, J. (1980). The temperature dependence of beam damage to biological samples in the scanning transmission electron microscope (STEM).Proc. 7th. Eur. Congr. Elec. Microsc., The Haag, vol. 2 (ed. Brederoo, P. and Boom, G.), pp. 640641. Leiden: The 7th. Eur. Congr. Foundation.
Fujiyoshi, Y., Uyeda, N., Yamajishi, H., Morikawa, K., Mizusaki, T., Aoki, Y., Kihara, H. & Harada, Y. (1986). Biological marcromolecules observed with high resolution cryo-electron microscope.In Proc. XIth Int. Cong. Elec. Microsc., Kyoto, vol. III (ed. Imura, T., Maruse, S. and Susuki, T.), pp. 18291832. Tokyo: Jap. Soc. Elec. Microsc.
Fukami, A. & Adachi, K. (1965). A new method of preparation of a self-perforated micro-plastic grid and its application. J. Elec. Microsc. (Japan) 14, 112118.
Fukami, A., Adachi, K. & Katoh, M. (1972). Micro grid techniques (continued) and their contribution to specimen preparation techniques for high resolution work. J. Elec. Microsc. (Japan) 21, 99108.
Fuller, S. D. (1987). The T = 4 envelope of Sindbis virus is organized by interactions with a complementary T = 3 capsid. Cell 48, 923934.
Fuller, S. D. & Argos, P. (1987). Is Sindbis a simple picornavirus with an evelope? EMBO J. 26, 15031511.
Geiger, A., Mausbach, P. & Schnitker, J. (1986). Computer simulation study of the hydrogen-bond network in metastable water. In Water and Aqueous Solutions (ed. Neilson, G. W. and Enderby, J. E.), pp. 3140. Bristol: Adam Hilger.
De Gennes, P. G. (1984). Comment s'étale une goutte. Pour la Science 79, 8896.
De Gennes, P. G. (1985). Wetting: statistics and dynamics. Rev. mod. Phys. (USA) 57, 827863.
Ghormley, J. A. & Hochanadel, C. J. (1971). Amorphous ice: density and reflectivity. Science 171, 6264.
Glaeser, R. M. (1971). Limitation to significant information in biological electron microscopy as a result of radiation damage. J. Ultrastruct. Res. 36, 466482.
Glaeser, R. M. (1975). Radiation damage and biological electron microscopy. In Physical Aspects of Electron Microscopy and Microbeam Analysis (ed. Siegel, B. M. and Bearman, D. R.), p. 205. New York: Wiley.
Glaeser, R. M. & Taylor, K. A. (1978). Radiation damage relative to transmission electron microscopy of biological specimens at low temperature: a review. J. Microsc. 112, 127138.
Griffiths, G., McDowall, A. W., Back, R. & Dubochet, J. (1984). On the preparation of cryosections for immunocrytochemistry. J. Ultrastruct. Res. 89, 6578.
Griffiths, G., Simons, K., Warren, G. & Tokuyasu, K. T. (1983). Immunoelectron microscopy using thin, frozen sections: application to studies of the intracellular transport of Semliki forest virus spike glycoproteins. Meth. Enzymol. 96, 435450.
Gupta, B. L. & Hall, T. A. (1981). The X-ray microanalysis of frozen-hydrated sections in scanning electron microscopy: an evaluation. Tissue Cell 13, 623643.
Hahn, M. (1980). Properties of commerical films for electron microscopy. In Electron Microscopy and Molecular Dimensions (ed. Baumesiter, W. and Vogell, W.), pp. 200207. Heidelberg: Springer-Verlag.
Hall, T. A. & Gupta, B. L. (1974). Beam-induced loss of organic mass under electron microscope conditions. J. Microsc. 100, 177188.
Handley, D. A., Alexander, J. T. & Chien, S. (1981). The design and use of a simple device for rapid quench-freezing of biological samples. J. Microsc. 121, 273282.
v. Harreveld, A., Trubatch, J. & Steiner, J. (1974). Rapid freezing and electron microscopy for the arrest of physiological processes, J. Microsc. 100, 189198.
Hart, R. K., Kassner, T. F. & Maurin, J. K. (1970). Contamination of surfaces during high-energy electron radiation. Phil. Mag. 21, 453467.
Hayat, M. A. (1970). Principles and Techniques of Electron Microscopy. Biological Applications, vol. 1. London: Van Nostrand Reinhold.
Hayward, S. B., Grano, D. A., Glaeser, R. M. & Fisher, K. A. (1978). Molecular orientation of bacteriorhodopsin within the purple membrane of halobacterium halobium.Proc. natn. Acad. Sci. U.S.A. 75, 43204324.
van Heel, M. & Frank, J. (1981). Use of multivariate statistics in image analysing the images of biological macromolecules. Ultramicroscopy. 6, 187194.
Heide, H. G. (1982 a). On the irradiation of organic samples in the vicinity of ice. Ultramicroscopy. 7, 299300.
Heide, H. G. (1982 b). Design and operation of cold stages. Ultramicroscopy. 10, 125154.
Heide, H. G. & Grund, S. (1974). Eine Tiefkühlkette zum Überführen von wasserhaltigen biologischen Objekten im Elektronenmikroskop. J. Ultrastruct. Res. 48, 259268.
Heide, H. G. & Zeitler, E. (1985). The physical behaviour of solid water at low temperatures and the embedding of electron microscopical specimens. Ultramicroscopy 16, 151160.
Heidenreich, R. R. (1964). Fundamentals of Transmission Electron Microscopy. New York: Interscience Publ.
Heuser, J. E., Reese, T. S., Dennis, M. J., Jan, Y., Jan, L. & Evans, L. (1979). Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J. Cell Biol. 81, 275300.
Hiromi, K. (1979). Kinetics of Fast Enzyme Reactions. Theory and Practice. Kodansha Scientific Books. New York: Wiley.
Hobbs, P. V. (1974). Ice Physics. Oxford: Clarendon Press.
Homo, J.-Cl., Booy, F., Labouesse, P., Lepault, J. & Dubochet, J. (1984). Improved anticontaminator for cryo-electron microscopy with Philips EM 400. J. Microscopy 136, 337340.
Hutchinson, T. E., Bacaner, M., Brodhurst, J. & Lilley, J. (1974). Electron microscopy of frozen biological tissue. Rev. scient. Instrum. 45, 252255.
Huttermann, J. (1982 a). Physical mechanisms of electron interaction with organic solids. Ultramicroscopy. 10, 714.
Huttermann, J. (1982 b). Solid-state radiation chemistry of DNA and its constituents. Ultramicroscopy. 10, 2540.
International Expermental Study Group (1986). Cryoprotection in electron microscopy. J. Microsc. 141, 385391.
Isaacson, M. S. (1977). Specimen damage in the electron microscope. In Principles and Techniques of Electron Microscopy. Biological Applications, vol. 7 (ed. Hayat, M. A.), pp. 178. New York: Van Nostrand Reinhold.
Jaffe, J. S. & Glaeser, R. M. (1984). Preparation of frozen-hydrated specimens for high resolution electron microscopy. Ultramicroscopy 13, 373378.
Jakubowski, U. (1985). Can heat pipes solve the problems of drift and vibration of cryoholders? Ultramicroscopy. 17, 379382.
Jeng, T.-W. & Chiu, W. (1984). Quantitative assessment of radiation damage in a thin protein crystal. J. Microsc. 136, 3544.
Jesior, J.-Cl. (1987). How to avoid compression. II. The influence of sectioning conditions. J. Ultrastruct. Res. (In the Press.)
Johari, G. P. (1977). On the heat capacity, entropy and glass transition of vitreous ice. Phil. Mag. 35 (4), 10771090.
Kellenberger, E. (1987). The response of biological macromolecules and supramolecular structures to the physics of cryo-specimen preparation. In Cryotechniques in Biological Electron Microscopy (ed. Steinbrecht, R. A. and Zierold, K., pp. 3563. Heidelberg: Springer-Verlag.
Kellenberger, E., Carlemalm, E. & Villiger, W. (1986 b). Physics of the preparation and observation of specimens that involve cryoprocedure. In Science of Biological Specimen Preparation (ed. Müller, M., Becker, R., Boyde, A. and Wolosewick, J.), pp. 120. Chicago: SEM Inc., AMF O'Hare.
Kellenberger, E., Häner, M. & Wurtz, M. (1982). The wrapping phenomenon in air-dried and negatively stained preparations. Ultramicroscopy. 9, 139150.
Kellenberger, E. & Kistler, J. (1979). The physics of specimen preparation. In Advances in Structure Research by Diffraction Methods, vol. VIII (ed. Hoppe, W. and Mason, R.), pp. 4979. Wiesbaden: Friedr. Vieweg.
Kistler, J. & Kellenberger, E. (1977). Collapse phenomena in freeze-drying. J. Ultrastruct. Res. 59, 7075.
Kleinschmidt, A. K. & Zahn, R. K. (1959). Über Deoxyribonucleinsäure-Molekulen in Protein-Mischfilmen. Z. Naturf. 14b, 770779.
Knapek, E. & Dubochet, J. (1980). Beam damage to organic crystals is considerably reduced in cryo-electron microscopy. J. molec. Biol. 141, 147161.
Kobayashi, K. & Sakaoku, K. (1965). Irradiation changes in organic polymers at various acceleration voltages. Lab. Investigation 14, 10971114.
König, H. (1943). Eine kubische Eismodification. Z. Krisṫallogr. 105, 279286.
Kohl, H., Rose, H. & Schnabl, H. (1981). Dose-rate effect at low temperatures in FBEM and STEM due to object-heating. Optik 58, 1124.
Langer, J. S. (1980). Growth of ice crystals. Rev. mod. Phys. 52, 128.
Lamvik, M. K., Kopf, D. A. & Robertson, J. D. (1983). Radiation damage in L-valine at liquid helium temperature. Nature 301, 332334.
Leisegang, S. (1954). Zur Erwärmung elektronenmikroskopischer Objekte bei kleinem Strahlquerschnitt.In. Proc. 3rd Int. Conf. Elec. Microsc., London, pp. 176188.
Lenz, S. (1954). Zur Streuung mittelschneller Elektronen in kleinste Winkel. Z. Naturf. 9 a, 185204.
Lepault, J. (1985). Cryo-electron microscopy of helical particles TMV and T4 polyheads. J. Microsc. 140, 7380.
Lepault, J., Booy, F. P. & Dubochet, J. (1983 a). Electron microscopy of frozen biological suspensions. J. Microsc. 129, 89102.
Lepault, J. & Dubochet, J. (1980). Freezing, fracturing and etching artefacts in particulate suspensions. J. Ultrastruct. Res. 72, 223233.
Lepault, J. & Dubochet, J. (1986 a). Electron microscopy of frozen hydrated specimens: preparation and characteristics. In Meth. Enzymol. 127, 719730.
Lepault, J. & Dubochet, J. (1986 b). Beam damage and frozen-hydrated specimens.In Proc. XIth Int. Cong. Elec. Microsc., Kyoto, vol. 1 (ed. Imura, T., Maruse, S. and Suzuki, T.), pp. 2528. Tokyo: Japanese Society for Electron Microscopy.
Lepault, J., Dubochet, J., Baschong, W. & Kellenberger, E. (1987). Organization of double-stranded DNA in bacteriophages: a study by cryo-electron microscopy of vitrified samples. EMBO J. 6, 15071512.
Lepault, J., Dubochet, J., Dietrich, I., Knapek, E. & Zeitler, E. (1983 b). Amendment to: Electron beam damage to organic specimens at liquid helium temperature. J. molec. Biol. 163, 511.
Lepault, J., Freeman, R. & Dubochet, J. (1983 c). Electron beam induced ‘vitrified ice’. J. Microsc. 132, RP3–RP4.
Lepault, J. & Leonard, K. (1985). Three-dimensional structure of unstained frozen-hydrated extended tails of bacteriophage T4. J. molec. Biol. 182, 431441.
Lepault, J., Pattus, F. & Martin, N. (1985). Cryo-electron microscopy of artificial biological membranes. Biochim. biophys. Acta 820, 315318.
Lepault, J. & Pitt, T. (1984). Projected structure of unstained, frozen-hydrated T-layer of Bacillus brevis. EMBO J. 3, 101105.
Lickfeld, K. G. (1985). Ein Beitrag zur Frage welche Kräfte und Faktoren Dünnschneiden bewirken. J. Ultrastruct. Res. 93, 101115.
Luyet, B. J. & Gehenio, P. M. (1940). Life and Death at Low Temperatures. Normandy, Missouri: Biodynamica.
Mackenzie, A. P. (1977). Non equilibrium freezing behaviour of aqueous systems. Phil. Trans. R. Soc. Lond. B 278, 167189.
Maddox, J. (1983). Snowflakes are far from simple. Nature 306, 13.
Mandelkow, E.-M., Rapp, R. & Mandelkow, E. (1986). Microtubule structure studied by quick freezing: cryo-electron microscopy and freeze fracture. J. Microsc. 141, 361373.
Mayer, E. (1985). Vitrification of pure liquid water. J. Microsc. 140, 315.
Mayer, E. & Brüggeller, P. (1982). Vitrification of pure liquid water by high pressure jet freezing. Nature 298, 715718.
Mayer, E. & Brüggeller, P. (1983). Devitrification of glassy water. Evidence for a discontinuity of states. J. phys. Chem. 87, 47444749.
Mayer, E. & Hallbrucker, A. (1987). Cubic ice from liquid water. Nature 325, 601602.
Mazur, P. (1970). Cryobiology: the freezing of biological systems. Science 168, 939949.
Mazur, P. (1984). Freezing of living cells: Mechanisms and implications. Am. J. Physiol. 247, C125–C142.
McDowall, A. W., Chang, J.-J., Freeman, R., Lepault, J., Walter, C. A. & Dubochet, J. (1983). Electron microscopy of frozen hydrated sections of vitreous ice and vitrified biological samples. J. Microsc. 131, 19.
McDowall, A. W., Hofmann, W., Lepault, J., Adrian, M. & Dubochet, J. (1984). Cryo-electron microscopy of vitrified insect flight muscle. J. molec. Biol. 178, 105111.
McDowall, A. W., Smith, J. M. & Dubochet, J. (1986). Cryo-electron microscopy of vitrified chromosomes in situ. EMBO J. 5, 13951402.
Menco, B. P. M. (1986). A survey of ultra-rapid cryofixation methods with particular emphasis on applications to freeze-fracturing, freeze-etching, and freeze-substitution. J. Elec. Microsc. Techn. 4, 177240.
Milligan, R. & Flicker, J. (1987). J. Cell Biol. 105, 2939.
Mishima, O., Calvert, L. D. & Whalley, E. (1984). ‘Melting ice’ I at 77K and 10Kbar: a new method of making amorphous solids. Nature 310, 393395.
Moor, H. (1987). Theory and practice of high pressure freezing. In Cryotechniques in Biological Electron Microscopy (ed. Steinbrecht, A. and Zierold, K.). Heidelberg: Springer Verlag, (in the Press).
Müller, M., Meister, N. & Moor, H. (1980). Freezing in a propane jet and its application in freeze-fracturing. Mikroskopie (Vienna) 36, 129140.
Müller, M. & Moor, H. (1984). Cryofixation of thick specimens by high pressure freezing. In Science of Biological Specimen Preparation, vol. 2 (ed. Revel, J.-P., Barnard, T. and Haggis, G. H.), pp. 131138. Chicago: SEM Inc., AMF O'Hare.
Narten, A. H. & Levy, H. A. (1969). Observed diffraction pattern and proposed models of liquid water. Science 165, 447454.
Neilson, G. W. & Enderby, J. E. (eds.) (1986). Water and Aqueous Solutions. Bristol & Boston: A. Hilger.
Nittmann, J. & Stanley, H. E. (1986). Tip splitting without interfacial tension and dendritic growth patterns arising from molecular anisotropy. Nature 321, 663668.
Parsegian, V. A. (1975). Long range van der Waals forces. In Physical Chemistry: Enriching Topics from Colloid and Surface Science (ed. Olphen, H. V. & Mysels, K. J.), pp. 2772. Série IUPAC.
Parsons, D. F. (1974). Structure of wet specimens in electron microscopy. Science 186, 407414.
Pauling, L. (1970). General Chemistry, 3rd ed. San Francisco: Freeman.
Plattner, H. & Bachmann, L. (1982). Cryofixation: A tool in biological ultrastructural research. Int. Rev. Cytol. 79, 237304.
Plattner, H. & Knoll, G. (1984). Cryofixation of biological materials for electron microscopy by the methods of spray-, sandwich-, cryogen-jet- and sandwich-cryogen-jet-freezing: A comparison of techniques. In Science of Biological Specimen Preparation, vol. 2 (ed. Revel, J.-P., Barnard, T. and Haggis, G. H.), pp. 139146. Chicago: SEM Inc., AMF O'Hare.
Polge, C., Smith, A. U. & Parkes, A. S. (1949). Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164, 666667.
Polian, A. & Grimsditch, M. (1984). New high pressure phase of H2O: Ice X. Phys. Rev. Lett. 52, 13121314.
Rachel, R., Jakubowski, U., Tietz, H., Hegerl, R. & Baumeister, W. (1986). Projected structure of the surface protein of deinococcus radiodurans determined to 8 Å resolution by cryomicroscopy. Ultramicroscopy, 20, 305316.
Rahman, A., & Stillinger, F. H. (1971). Molecular dynamics study of liquid water. J. chem. Phys. 55, 33363359.
Rasmussen, D. H. (1982). Ice formation in aqueous systems. J. Microsc. 128, 167174.
Rasmussen, D. H. & Mackenzie, A. P. (1973). Clustering in supercooled water. J. chem. Phys. 59, 50035013.
Reimer, L. (1975). Review of the radiation damage problem of organic specimens in electron microscopy. In Physical Aspects of Electron Microscopy and Microbeam Analysis (ed. Siegel, B. M. and Beaman, D. R.), pp. 231245. New York: Wiley.
Reimer, L. (1984). Electron microscopy. Heidelberg: Springer Verlag.
Riehle, U. (1968). Schnellgefrieren organischer Präparate für die Elektronen-Mikroskopie. Chemie-Ing. Techn. 40, 213218.
Riehle, U. & Hochli, M. (1973). The theory and technique of high pressure freezing. In Freeze-Etching Techniques and Applications (ed. Benedetti, E. L. and Farvard, P.), pp. 3161. Paris: Société Française de Microscopie Electronique.
Robards, A. W. & Sleytr, U. B. (1985). Low Temperature Methods in Biological Electron Microscopy. New York: Elsevier.
Roberts, I. M. (1975). Tungstein coating – a method of improving glass microtome knives for cutting ultrathin sections. J. Microsc. 103, 113119.
Sander, C. M. (1986). Fractal growth processes. Nature 322, 789793.
Saxton, W. O. (1978). Computer Techniques for Image Processing in Electron Microscopy. Academic Press.
Sceats, M. G. & Rice, S. A. (1982). Amorphous solid water and its relationship to liquid water: A random network model for water. In Water: A Comprehensive Treatise (ed. Franks, F.), pp. 83214. New York: Plenum Press.
Schäfer, L., Yates, A. C., Bonham, R. A. (1971). New values for the partial wave electron scattering factor for the elements 1 ≤ Z ≤ 57 and 72 ≤ Z ≤ 90 for incident electron energies of 10, 40, 70 and 100 keV. J. chem. Phys. 55, 30553056.
Scherzer, O. (1949). The theoretical resolution limit of the electron microscope, J. appl. Phys. 20, 2029.
Siegel, G. (1972). The influence of very low temperature on the radiation damage of organic crystals irradiated by 100 kV electrons. Z. Naturf. A 27, 325332.
Sitte, H., Edelmann, L. & Neumann, K. (1987). Cryofixation without pretreatment at ambient pressure. In Cryotechniques in Biological Electron Microscopy (ed. Steinbrecht, R. A. and Zierold, K., pp. 87113). Heidelberg: Springer Verlag.
Sitte, H. & Neumann, K. (1983). Beitrag 1. 1. 2. In Methodensammlung der Elektronenmikroskopie (ed. Schimmel, G. and Vogell, W.), pp. 1248. Stuttgart: Wiss. Verlag GmbH.
Sitte, H., Neumann, K., Hässig, H., Kleber, H. & Kappl, G. (1980). FC4 cryochamber for Reichert OmU4-ultramicrotome Ultracut.In Proc. 7th Eur. Congr. Elec. Microsc., The Hague, vol. 2 (ed. Brederoo, P. and Boom, G.), pp. 540541. Leiden: Eur. Congr. Fundation.
Sleytr, U. B. & Robards, A. W. (1977). Plastic deformation during freezing-cleavage: a review. J. Microsc. 110, 125.
Stanley, H. E. & Teixeira, J. (1980). Interpretation of the unusual behaviour of H2O and D2O at low temperatures: tests of a percolation model. J. chem. Phys. 73, 34043422.
Stewart, M. & Vigers, G. (1986). Electron microscopy of frozen-hydrated biological material. Nature 319, 631636.
Stillinger, F. H. (1980). Water revisited. Science 309, 451457.
Stillinger, F. H. & Weber, T. A. (1984). Packing structures and transitions in liquids and solids. Science 225, 983989.
Symons, M. C. R. (1982 a). Chemical aspects of electron beam interactions in the solid state. Ultramicroscopy 10, 1524.
Symons, M. C. R. (1982 b). The pre knock-on concept. Ultramicroscopy 10, 4144.
Talmon, Y. (1982). Thermal and radiation damage to frozen hydrated specimens. J. Microsc. 125, 227237.
Talmon, Y. (1984). Radiation damage to organic inclusions. Ultramicroscopy 14, 305316.
Talmon, Y., Adrian, M. & Dubochet, J. (1986). Electron beam damage to organic inclusions in vitreous, cubic and hexagonal ice. J. Microsc. 141, 375384.
Talmon, Y., Davis, H. T., Scriven, L. E., & Thomas, E. L. (1979). Mass loss and etching of frozen hydrated specimens. J. Microsc. 117, 321332.
Talmon, Y. & Thomas, E. L. (1977 a). Beam heating of a moderately thick cold stage specimen in the SEM/STEM. J. Microsc. III, 151164.
Talmon, Y. & Thomas, E. L. (1977 b). Temperature rise and sublimation of water from this frozen hydrated specimens in cold stage microscopy. Scanning Elec. Microsc. 1, 265272.
Talmon, Y. & Thomas, E. L. (1978). Electron beam heating temperature profiles in moderately thick cold stage STEM/SEM specimens. J. Microsc. 113, 6975.
Talmon, Y. & Thomas, E. L. (1979). Open system microthermometry – a technique for the measurement of local specimen temperature in the electron microscope. J. Mater. Sci. 14, 16471650.
Tanford, C. (1961). Physical Chemistry of Macromolecules. New York: Wiley.
Tatlock, G. J. (1982). Solid gases. Ultramicroscopy 10, 8796.
Taylor, K. A. (1978). Structure determination of frozen, hydrated, crystalline biological specimens. J. Microsc. 112, 15125.
Taylor, K. A. & Glaeser, R. M. (1974). Electron diffraction of frozen, hydrated protein crystals. Science 106, 1036–37.
Taylor, K. A. & Glaeser, R. M. (1976). Electron microscopy of frozen hydrated biological specimens. J. Ultrastruct. Res. 55, 448456.
Taylor, K. J., Chanzy, H. & Marchessault, R. H. (1975). Electron diffraction for hydrated crystalline biopolymers: Nigeran. J. molec. Biol. 92, 165167.
Teixeira, J., Stanley, E., Bottinga, Y. & Richet, P. (1983). Application of a percolation model to supercooled liquids with a tetrahedral structure. Bull. Minéral. 106, 99105.
Thornbury, W. & Mengers, P. E. (1957). An analysis of frozen section techniques. I. Sectioning of fresh-frozen tissues. J. Histochem. Cytochem. 5, 4752.
Tokuyasu, K. T. (1973). A technique for ultracryotomy of cell suspensions and tissues. J. Cell Biol. 57, 551565.
Tokuyasu, K. T. (1980). Immunochemistry of ultrathin frozen sections. Histochem. J. 12, 381403.
Tokuyasu, K. T. & Okamura, S. (1959). A new method for making glass knives for thin sectioning. J. Biophys. Biochem. Cytol. 6, 305308.
Trinick, J., Cooper, J., Seymour, J. & Egelman, E. H. (1986). Cryo-electron microscopy and three-dimensional reconstruction of actin filaments. J. Microsc. 141, 349360.
Typke, D. & Radermacher, M. (1982). Determination of the phase of complex atomic scattering amplitudes from light-optical diffractograms of electron microscope images. Ultramicroscopy 9, 131138.
Unwin, P. N. T. (1972). Electron microscopy of biological specimens by means of an electrostatic phase plate.Proc. R. Soc. London A 329, 327359.
Unwin, P. N. T. (1974). Electron microscopy of the stacked dish aggregate of Tobacco Mosaic Virus protein. II. The influence of electron irradiation on the stain distribution, J. molec. Biol. 87, 657670.
Unwin, P. N.T. & Henderson, R. (1975). Molecular structure determination by electron microscopy of unstained crystalline specimens. J. molec. Biol. 94, 425440.
Unwin, P. N.T. & Muguruma, J. (1971). Transmission electron microscopy of ice. J. appl. Phys. 42, 36403641.
Unwin, P. N.T. & Muguruma, J. (1972). Electron microscope observations on the defect structure of ice. Physica status solidi (a) 14, 207216.
Valentine, R. C. (1966). Response of photographic materials to electrons. In Advances in Optical and Electron Microscopy, vol. 1 (ed. Barer, R. and Cosslett, V. E.), pp. 180202. London: Academic Press.
Vigers, G. P. A., Crowther, R. A. & Pearse, B. M. F. (1986 a). Three-dimensional structure of clathrin cages in ice. EMBO J. 5, 529534.
Vigers, G. P. A., Crowther, R. A. & Pearse, B. M. F. (1986 b). Location of the 100kD–50kD accessory proteins in clathrin coats. EMBO J. 5, 20792085.
Vogel, R. H., Provencher, S. W., Von Bonsdorff, C.-H., Adrian, M. & Dubochet, J. (1986). Envelope structure of Semliki forest virus reconstructed from cryo-electron micrographs. Nature 320, 533535.
Wade, R. H. (1984). The temperature dependence of radiation damage in organic and biological material. Ultramicroscopy 14, 265270.
Wall, J., Bittner, J. W. & Hainfeld, J. (1977). Contamination at low temperature.In Proc. 35th Ann. EMSA Meeting, Boston, pp. 558559.
Wall, J., Isaacson, M. & Langmore, J. (1974). The collection of scattered electrons in dark-field electron microscopy. II. Inelastic scattering. Optik 39, 359374.
Wilson, D. (1979). Supercold: An Introduction to Low Temperature Technology, London & Boston: Faber & Faber.
Zierold, K. (1982). Preparation of biological cryosections for analytical electron microscopy. Ultramicroscopy 10, 4554.
Zierold, K. (1984). The morphology of ultrathin cryosections. Ultramicroscopy 14, 201209.
Zierold, K. (1987). Cryoultramicrotomy. In Cryotechniques in Biological Electron Microscopy (ed. Steinbrecht, A. and Zierold, K., pp. 132148). Heidelberg: Springer Verlag.

Cryo-electron microscopy of vitrified specimens

  • Jacques Dubochet (a1), Marc Adrian (a1), Jiin-Ju Chang (a1), Jean-Claude Homo (a1), Jean Lepault (a1), Alasdair W. McDowall (a1) and Patrick Schultz (a1)...


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