Skip to main content
SpringerLink
Log in

Cryoelectron Microscopy of Vitrified Specimens

  • Chapter
Cryotechniques in Biological Electron Microscopy

Abstract

Water is the most abundant component of biological material, but it is systematically excluded from conventional electron microscopy. This is because water evaporates rapidly under the vacuum conditions of an electron microscope. Cryoelectron microscopy has long been seen as a possible avenue to overcome this limitation, but until recently the direct observation of frozen-hydrated specimens was relatively unsuccessful because of a number of serious difficulties. These were, in particular, due to the absence of a good cryospecimen holder, the inherently low contrast of hydrated specimens and the structural damage due to ice crystals formed during freezing. As a consequence, the cryomethods which have flourished in electron microscopy during the last 20 years were not aimed at preserving the hydration of the specimen in the electron microscope. Freezing was only used as an aid to preparation. The objects ultimately observed in the electron microscope were dry and at room temperature. Such cryomethods have recently been reviewed in detail (Robards and Sleytr 1985).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Adrian M, Dubochet J, Lepault J, McDowall AW (1984) Cryo-electron microscopy of viruses. Nature (London) 308:32–36.

    Article  CAS  Google Scholar 

  2. Angell CA (1982) Supercooled water. In: Franks F (ed) Water: A comprehensive treatise, vol 7. Plenum, New York London, pp 1–81.

    Google Scholar 

  3. Brüggeller P, Mayer E (1980) Complete vitrification in pure liquid water and dilute aqueous solutions. Nature (London) 288:569–571.

    Article  Google Scholar 

  4. Chang J-J, McDowall AW, Lepault J, Freeman R, Walter CA, Dubochet J (1983) Freezing, sectioning and observation artefacts of frozen hydrated sections for electron microscopy. J Microsc (Oxford) 132:109–123.

    Article  Google Scholar 

  5. Chanzy H, Guizard C, Vuong R (1977) Electron diffraction on frozen hydrated polysaccharides. J Microsc (Oxford) 111:143–150.

    Article  CAS  Google Scholar 

  6. Chiu W (1986) Electron microscopy of frozen hydrated biological specimens. Annu Rev Biophys Chem 15:237–257.

    Article  CAS  Google Scholar 

  7. Chiu W, Downing KH, Dubochet J, Glaeser RM, Heide HG, Knapek E, Kopf DA, Lamvik MK, Lepault J, Robertson JD, Zeitler E, Zemlin F (International Experimental Study Group) (1986) Cryoprotection in electron microscopy. J Microsc (Oxford) 141:385–391.

    Article  Google Scholar 

  8. Douzou P (1977) Cryobiochemistry: an introduction. Academic Press, London New York.

    Google Scholar 

  9. Dubochet J, McDowall AW (1981) Vitrification of pure water for electron microscopy. J Microsc (Oxford) 124:RP3–4.

    Article  Google Scholar 

  10. Dubochet J, McDowall AW (1984a) Cryoultramicrotomy: study of ice crystals and freezing damage. In: Csanady A, Röhlich P, Szabo D (eds) Electron microscopy 1984, vol 2. 8th Eur Congr Electron Microsc Budapest, pp 1407–1410.

    Google Scholar 

  11. Dubochet J, McDowall AW (1984b) Frozen hydrated sections. In: Revel J-P, Barnard T, Haggis GH (eds) The science of biological specimen preparation. SEM, AMF O’Hare, IL 60666, pp 147-152.

    Google Scholar 

  12. Dubochet J, Chang J-J, Freeman R, Lepault J, McDowall AW (1982a) Frozen aqueous suspensions. Ultramicroscopy 10:55–62.

    Article  Google Scholar 

  13. Dubochet J, Lepault J, Freeman R, Berriman JA, Homo J-C (1982b) Electron microscopy of frozen water and aqueous solutions. J Microsc (Oxford) 128:219–237.

    Article  Google Scholar 

  14. Dubochet J, Groom M, Müller-Neuteboom S (1982c) The mounting of macromolecules for electron microscopy. In: Cosslett VE, Barer R (eds) Advances in optical and electron microscopy, vol 8. Academic Press, London New York, pp 107–135.

    Google Scholar 

  15. Dubochet J, McDowall AW, Menge B, Schmid EN, Lickfeld KG (1983) Electron microscopy of frozen-hydrated bacteria. J Bacteriol 155:381–390.

    PubMed  CAS  Google Scholar 

  16. Dubochet J, Adrian M, Lepault J, McDowall AW (1985) Cryo-electron microscopy of vitrified biological specimens. Trends Biochem Sci 10:143–146.

    Article  Google Scholar 

  17. Dubochet J, Adrian M, Schultz P, Oudet P (1986) Cryo-electron microscopy of vitrified SV40 minichromosomes. The liquid drop model. EMBO J 5:519–528.

    PubMed  CAS  Google Scholar 

  18. Dubochet J, Adrian M, Chang J-J, Lepault J, McDowall AW (1987) Cryo-electron microscopy of vitrified specimens. Q Rev Biophys (in press).

    Google Scholar 

  19. Echlin P (1971) The examination of biological material at low temperature. In: Johari O, Cerwin J (eds) Proc 4th Annual scanning electron microscope Symp, pt 1. IIT Press, Chicago, pp 227-232.

    Google Scholar 

  20. Erikson HP, Klug A (1970) The Fourier transform of an electron micrograph: effects of defocussing and aberrations, and implications for the use of underfocus contrast enhancement. Bunsenges Phys Chem 74:129–1137.

    Google Scholar 

  21. Fahy GM, Macarlane DR, Angell CA, Meryman HT (1984) Vitrification as an approach to cryopreservation. Cryobiology 21:407–426.

    Article  PubMed  CAS  Google Scholar 

  22. Fernández-Morán 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 85:689–713

    Article  PubMed  Google Scholar 

  23. Fernández-Morán H (1985) Cryo-electron microscopy and ultramicrotomy: Reminiscence and reflections. In: Advances in electronics and electron physics, suppl 16. Academic Press, London New York, pp 167–223.

    Google Scholar 

  24. Franks F (1982) The properties of aqueous solutions at subzero temperatures. In: Franks F (ed) Water: a comprehensive treatise, vol 7. Plenum, New York London, pp 215–338.

    Google Scholar 

  25. Frederik PM, Busing WM, Persson A (1984) Surface defects in thin cryosections. Scanning Electron Microsc 1984/I:433–443.

    Google Scholar 

  26. Fuller SD (1987) The T = 4 envelope of Sindhis virus is organised by interactions with a complementary T = 3 capsid. Cell 48:923–934.

    Article  PubMed  CAS  Google Scholar 

  27. Glaeser RM, Taylor KA (1978) Radiation damage relative to transmission electron microscopy of biological specimens at low temperature; a review. J Microsc (Oxford) 112:127–138.

    Article  CAS  Google Scholar 

  28. Griffiths G, McDowall AW, Back R, Dubochet J (1984) On the preparation of cryosections for immunocytochemistry. J Ultrastruct Res 29:65–78.

    Article  Google Scholar 

  29. Hiromi K (1979) Kinetics of fast enzyme reactions. Theory and practice. Kodansha scientific books. John Wiley & Sons, New York.

    Google Scholar 

  30. Hutchinson TE, Bacaner M, Brodhurst J, Lilley J (1974) Electron microscopy of frozen biological tissues. Rev Sci Instrum 45:252–255.

    Article  PubMed  CAS  Google Scholar 

  31. Hutchinson TE, Johnson DE, Macenzie AP (1978) Instrumentation for direct observation of frozen hydrated specimens in the electron microscope. Ultramicroscopy 3:315–324.

    Article  PubMed  CAS  Google Scholar 

  32. Jaffa JS, Glaeser RM (1984) Preparation of frozen-hydrated specimens for high resolution electron microscopy. Ultramicroscopy 13:373–378.

    Article  Google Scholar 

  33. Jeng T-W, Chiu W (1984) Quantitative assessment of radiation damage in a thin protein crystal. J Microsc (Oxford) 136:35–44.

    Article  CAS  Google Scholar 

  34. Johari GP (1977) On the heat capacity, entropy and “glass transition” of vitreous ice. Philos Mag 35:1077–1090.

    Article  CAS  Google Scholar 

  35. Kleinschmidt AK, Zahn RK (1959) Über Deoxyribonukleinsäure-Moleküle in Protein-Mischfilmen. Z Naturforsch 14b:770–779.

    CAS  Google Scholar 

  36. Lepault J (1985) Cryo-electron microscopy of helical particles TMV and T4 polyheads. J Microsc (Oxford) 140:73–80.

    Article  CAS  Google Scholar 

  37. Lepault J, Leonard K (1985) Three-dimensional structure of unstained frozen-hydrated extended tails of bacteriophage T4. J Mol Biol 182:431–441.

    Article  PubMed  CAS  Google Scholar 

  38. Lepault J, Pitt T (1984) Projected structure of unstained, frozen-hydrated T-layer of Bacillus brevis. EMBO J 3:101–105.

    PubMed  CAS  Google Scholar 

  39. Lepault J, Booy FP, Dubochet J (1983a) Electron microscopy of frozen biological suspensions. J Microsc (Oxford) 129:89–102.

    Article  CAS  Google Scholar 

  40. Lepault J, Freeman R, Dubochet J (1983b) Electron beam induced “vitrified ice”. J Microsc (Oxford) 132:RP3–RP4.

    Article  CAS  Google Scholar 

  41. Lepault J, Pattus F, Martin N (1985) Cryo-electron microscopy of artificial biological membranes. Biochim Biophys Acta 820:315–318.

    Article  CAS  Google Scholar 

  42. 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 6:1507–1512.

    PubMed  CAS  Google Scholar 

  43. Luyet BJ, Gehenio PM (1940) Life and death at low temperatures. Biodynamica, Normandy, Miss.

    Book  Google Scholar 

  44. MaeKenzie AP (1977) Non equilibrium freezing behaviour of aqueous systems. Philos Trans R Soc London Scr B278:167–189.

    Article  Google Scholar 

  45. Mandelkow E-M, Rapp R, Mandelkow E (1986) Microtubule structure studied by quick freezing: cryo-electron microscopy and freeze fracture. J Microsc (Oxford) 141:361–373.

    Article  CAS  Google Scholar 

  46. Mayer E (1985) Vitrification of pure liquid water. J Microsc (Oxford) 140:3–15.

    Article  CAS  Google Scholar 

  47. Mazur P (1970) Cryobiology: The freezing of biological systems. Science 168:939–949.

    Article  PubMed  CAS  Google Scholar 

  48. Mazur P (1984) Freezing of living cells, mechanisms and implications. Am J Physiol 247, 16:C125–C142.

    PubMed  CAS  Google Scholar 

  49. McDowall AW, Chang J-J, Freeman R, Lepault J, Walter CA, Dubochet J (1983) Electron microscopy on frozen hydrated sections of vitreous ice and vitrified biological samples. J Microsc (Oxford) 131:1–9.

    Article  CAS  Google Scholar 

  50. McDowall AW, Hofmann W, Lepault J, Adrian M, Dubochet J (1984) Cryo-electron microscopy of vitrified insect flight muscle. J Mol Biol 178:105–111.

    Article  PubMed  CAS  Google Scholar 

  51. McDowall AW, Smith JM, Dubochet J (1986) Cryo-electron microscopy of vitrified chromosomes in situ. EMBO J 5:1395–1402.

    PubMed  CAS  Google Scholar 

  52. Milligan RA, Flecker PF (1986) Three-dimensional reconstruction of decorated thin filaments in a frozen hydrated state. Biophys J 49:220.

    Google Scholar 

  53. Milligan RA, Brisson A, Unwin PNT (1984) Molecular structure determination of crystalline specimens in frozen aqueous solutions. Ultramicroscopy 13:1–10.

    Article  PubMed  CAS  Google Scholar 

  54. Moreton RB, Echlin P, Gupta BL, Hall TA, Weis Fogh T (1974) Preparation of frozen hydrated tissue sections for X-ray microanalysis in the scanning electron microscope. Nature (London) 247:113–115.

    Article  CAS  Google Scholar 

  55. Parsons DF (1974) Structure of wet specimens in electron microscopy. Science 186:407–414.

    Article  PubMed  CAS  Google Scholar 

  56. Rall WF, Fahy GM (1985) Ice-free cryopreservation of mouse embryos at-196 °C by vitrification. Nature (London) 313:573–575.

    Article  CAS  Google Scholar 

  57. Rasmussen DH (1982) Ice formation in aqueous systems. J Microsc (Oxford) 128:167–174.

    Article  CAS  Google Scholar 

  58. Robards AW, Sleytr UB (1985) Low temperature methods in biological electron microscopy. In: Glauert AM (ed) Practical methods in electron microscopy, vol 10. Elsevier, Amsterdam.

    Google Scholar 

  59. Stewart M, Vigers G (1986) Electron microscopy of frozen-hydrated biological material. Nature (London) 319:631–636.

    Article  CAS  Google Scholar 

  60. Takahashi T, Hirsh A, Erbe EF, Bross JB, Steere RL, Williams RJ (1986) Vitrification of human monocytes. Cryobiology 23:103–115.

    Article  PubMed  CAS  Google Scholar 

  61. Talmon Y (1982) Thermal and radiation damage to frozen hydrated specimens. J Microsc (Oxford) 125:227–237.

    Article  Google Scholar 

  62. Talmon Y (1984) Radiation damage to organic inclusions in ice. Ultramicroscopy 14:305–316.

    Article  CAS  Google Scholar 

  63. Talmon Y, Adrian M, Dubochet J (1986) Electron beam damage to organic inclusions in vitreous, cubic and hexagonal ice. J Microsc (Oxford) 141:375–384.

    Article  CAS  Google Scholar 

  64. Taylor KA, Glaeser RM (1975) Electron diffraction of frozen, hydrated protein crystals. Science 186:1036–1037.

    Article  Google Scholar 

  65. Taylor KA, Glaeser RM (1976) Electron microscopy of frozen hydrated biological specimens. J Ultrastruct Res 55:448–456.

    Article  PubMed  CAS  Google Scholar 

  66. Tokuyasu KT (1973) A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol 57:551–563.

    Article  PubMed  CAS  Google Scholar 

  67. Tokuyasu KT (1980) Immunochemistry on ultrathin frozen sections. Histochem J 12:381–403.

    Article  PubMed  CAS  Google Scholar 

  68. Trinnick J, Cooper J, Seymour J, Egelman EH (1986) Cryo-electron microscopy and three-dimensional reconstruction of actin filaments. J Microsc (Oxford) 141:349–360.

    Article  Google Scholar 

  69. Unwin PNT, Ennis PD (1984) Two configurations of a channel-forming membrane protein. Nature (London) 307:609–613.

    Article  CAS  Google Scholar 

  70. Unwin PNT, Henderson R (1975) Molecular structure determination by electron microscopy of unstained crystalline specimens. J Mol Biol 94:425–440.

    Article  PubMed  CAS  Google Scholar 

  71. Unwin PNT, Muguruma J (1971) Transmission electron microscopy of ice. J Appl Phys 42:3640–3641.

    Article  CAS  Google Scholar 

  72. Vigers GPA, Crowther RA, Pearse BMF (1986a) Three dimensional structure of clathrin cages in ice. EMBO J 5:529–534.

    PubMed  CAS  Google Scholar 

  73. Vigers GPA, Crowther RA, Pearse BMF (1986b) Location of the 100kD-50kD accessory proteins in clathrin coats. EMBO J 5:2079–2085.

    PubMed  CAS  Google Scholar 

  74. Vogel RH, Provencher SW, Bonsdorff von C-H, Adrian M, Dubochet J (1986) Envelope structure of Semliki Forest Virus reconstructed from cryo-electron micrographs. Nature (London) 320:533–535.

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Jiin-Ju Chang

    Present address: Institute of Biophysics, Academia Sinica, Peking, China

Authors and Affiliations

  1. European Molecular Biology Laboratory, Meyerhofstr. 1, Postfach 102209, D-6900, Heidelberg, Germany

    Alasdair W. McDowall

Authors
  1. Jacques Dubochet
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marc Adrian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jiin-Ju Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jean Lepault
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alasdair W. McDowall
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Max-Planck-Institut für Verhaltensphysiologie, D-8131, Seewiesen, Germany

    Rudolf Alexander Steinbrecht

  2. Max-Planck-Institut für Systemphysiologie, Rheinlanddamm 201, D-4600, Dortmund, Germany

    Karl Zierold

Rights and permissions

Reprints and permissions

Copyright information

© 1987 Springer-Verlag Berlin Heidelberg

About this chapter

Cite this chapter

Dubochet, J., Adrian, M., Chang, JJ., Lepault, J., McDowall, A.W. (1987). Cryoelectron Microscopy of Vitrified Specimens. In: Steinbrecht, R.A., Zierold, K. (eds) Cryotechniques in Biological Electron Microscopy. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-72815-0_5

Download citation

  • DOI: https://doi.org/10.1007/978-3-642-72815-0_5

  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-642-72817-4

  • Online ISBN: 978-3-642-72815-0

  • eBook Packages: Springer Book Archive

Publish with us

Policies and ethics

Search

Navigation