The Nucleus pp 425-439 | Cite as

Cryoelectron Microscopy of Vitreous Sections: A Step Further Towards the Native State

  • Cedric Bouchet-Marquis
  • Stanislav Fakan
Part of the Methods in Molecular Biology book series (MIMB, volume 464)


Nuclear architecture has been investigated intensively by various electron microscopy (EM) methods. Most of these require chemical fixation of the sample, although cryofixation has also been used in combination with cryosubstitution and resin embedding. This approach allowed one to considerably increase the knowledge about the structural features of different nuclear domains and their involvement in nuclear functions. Cryoelectron microscopy of vitreous sections (CEMOVIS) has added a new dimension to the ultrastructural analysis of the cell nucleus, especially thanks to the possibility of observing the specimen in its hydrated state. In this way one can analyse, at high resolution, cellular structures as close as possible to their native state. In this chapter we describe in detail the different steps of the CEMOVIS method, which should allow an electron microscopist to perform cryosectioning and cryoelectron microscopy of vitrified biological material.


Cell nucleus Cryoelectron microscopy Cryofixation Vitrified sections Cryoelectron microscopy of vitreous sections CEMOVIS 


  1. 1.
    Monneron, A. and Bernhard, W. (1969) Fine structural organization of the interphase nucleus in some. mammalian cells. J. Ultrastruct. Res. 27, 266-288PubMedCrossRefGoogle Scholar
  2. 2.
    Fakan, S. (2004) The functional architecture of the nucleus as analysed by ultrastructural cytochemistry. Histochem. Cell Biol. 122, 83-93PubMedCrossRefGoogle Scholar
  3. 3.
    Tokuyasu, K. T. (1973) A technique for ultracryotomy of cell suspensions and tissues. J. Cell Biol. 57, 551-565PubMedCrossRefGoogle Scholar
  4. 4.
    McDowall, A. W., Chang, J. J., Freeman, R., Lepault, J., Walter, C. A., and Dubochet, J. (1983) Electron microscopy of frozen hydrated sections of vitreous ice and vitrified biological samples. J. Microsc. 131, 1-9PubMedCrossRefGoogle Scholar
  5. 5.
    McDowall, A. W., Hofmann, W., Lepault, J., Adrian, M., and Dubochet, J. (1984) Cryoelectron microscopy of vitrified insect flight muscle. J. Mol. Biol. 178, 105-111PubMedCrossRefGoogle Scholar
  6. 6.
    McDowall, A. W., Smith, J. M., and Dubochet, J. (1986) Cryo-electron microscopy of vitrified chromosomes in situ. EMBO J. 5, 1395-1402PubMedGoogle Scholar
  7. 7.
    Sartori Blanc, N., Senn, A., Leforestier, A., Livolant, F., and Dubochet, J. (2001) DNA in human and stallion spermatozoa forms local hexagonal packing with twist and many defects J. Struct. Biol. 134, 76-81CrossRefGoogle Scholar
  8. 8.
    Al-Amoudi, A., Chang, J. J., Leforestier, A., McDowall, A., Salamin, L. M., Norlen, L. P., Richter, K., Blanc, N. S., Studer, D., and Dubochet, J. (2004) Cryoelectron microscopy of vitreous sections. EMBO J. 23, 3583-3588PubMedCrossRefGoogle Scholar
  9. 9.
    Zuber, B., Nikonenko, I., Klauser, P., Muller, D., and Dubochet, J. (2005) The mammalian central nervous synaptic cleft contains a high density of periodically organized complexes. Proc. Natl. Acad. Sci. USA 102, 19192-19197PubMedCrossRefGoogle Scholar
  10. 10.
    Eltsov, M. and Dubochet, J. (2005) Fine structure of the Deinococcus radiodurans nucleoid revealed by cryoelectron microscopy of vitreous sections. J. Bacteriol. 187, 8047-8054PubMedCrossRefGoogle Scholar
  11. 11.
    Bouchet-Marquis, C., Dubochet, J., and Fakan, S. (2006) Cryoelectron microscopy of vitrified sections: a new challenge for the analysis of functional nuclear architecture. Histochem. Cell Biol. 125, 43-51PubMedCrossRefGoogle Scholar
  12. 12.
    Dubochet, J. and Sartori Blanc, N. (2001) The cell in absence of aggregation artifacts. Micron 32, 91-99PubMedCrossRefGoogle Scholar
  13. 13.
    Studer, D., Graber, W., Al-Amoudi, A., and Eggli, P. (2001) A new approach for cryofixation by high-pressure freezing. J. Microsc. 203, 285-294PubMedCrossRefGoogle Scholar
  14. 14.
    Eisenberg, D. and Kauzmann, W. (1969) The Structure and Properties of Water. Oxford University Press, OxfordGoogle Scholar
  15. 15.
    Franks, F. (1982) Water: A comprehensive treatise. In: Water and aqueous solutions at subzero temperatures (Franks, F., Ed.), Plenum Press, New York, pp. 215-338Google Scholar
  16. 16.
    Dubochet, J., Adrian, M., Chang, J. J., Homo, J. C., Lepault, J., McDowall, A. W., and Schultz, P. (1988) Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21, 129-228PubMedCrossRefGoogle Scholar
  17. 17.
    Dubochet, J., Al-Amoudi, A., Bouchet-Marquis, C., Eltsov, M., and Zuber, B. (in press) CEMOVIS. Cryo-electron microscopy of vitreous sections. In: Modern Cryopreparation methods for electron microscopy (Cavalier, A., Humbel, B. M., and Spehner, A., eds.), CRCGoogle Scholar
  18. 18.
    Al-Amoudi, A., Studer, D., and Dubochet, J. (2005) Cutting artefacts and cutting process in vitreous sections for cryo-electron microscopy. J. Struct. Biol. 150, 109-121PubMedCrossRefGoogle Scholar
  19. 19.
    Al-Amoudi, A., Dubochet, J., Gnaegi, H., Luthi, W., and Studer, D. (2003) An oscillating cryoknife reduces cutting induced deformation of vitreous ultrathin sections. J. Microsc. 212, 26-33PubMedCrossRefGoogle Scholar
  20. 20.
    Sartori Blanc, N., Studer, D., Ruhl, K., and Dubochet, J. (1998) Electron beam-induced changes in vitreous sections of biological samples. J Microsc 192, 194-201PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Cedric Bouchet-Marquis
    • 1
  • Stanislav Fakan
    • 1
  1. 1.Centre of Electron MicroscopyUniversity of LausanneLausanneSwitzerland

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