Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy (original) (raw)

Nature Methods volume 4, pages 215–217 (2007)Cite this article

Abstract

Cryo-electron microscopy can provide high-resolution structural information about cells and organelles in the nearly native, frozen-hydrated state. Applicability, however, is limited by difficulties encountered in preparing suitably thin, vitreously frozen biological specimens. We demonstrate, by cryo-electron tomography of Escherichia coli cells, that a focused ion beam (FIB) can be used to thin whole frozen-hydrated cells in a convenient and essentially artifact-free way.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 12 print issues and online access

$259.00 per year

only $21.58 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

References

  1. Sali, A., Glaeser, R., Earnest, T. & Baumeister, W. Nature 422, 216–225 (2003).
    Article CAS Google Scholar
  2. Nicastro, D., Frangakis, A.S., Typke, D. & Baumeister, W. J. Struct. Biol. 129, 48–56 (2000).
    Article CAS Google Scholar
  3. Koster, A.J. et al. J. Struct. Biol. 120, 276–308 (1997).
    Article CAS Google Scholar
  4. Medalia, O. et al. Science 298, 1209–1213 (2002).
    Article CAS Google Scholar
  5. McDowall, A.W. et al. J. Microsc. 131, 1–10 (1983).
    Article CAS Google Scholar
  6. Al-Amoudi, A., Studer, D. & Dubochet, J. J. Struct. Biol. 150, 109–121 (2005).
    Article CAS Google Scholar
  7. Hsieh, C.-E., Leith, A., Mannella, C.A., Frank, J. & Marko, M. J. Struct. Biol. 153, 1–13 (2006).
    Article CAS Google Scholar
  8. Gianuzzi, L.A. & Stevie, F.A. (eds.) Introduction to Focused Ion Beams (Springer, New York, 2005).
    Book Google Scholar
  9. Mulders, H. GIT Imaging and Microscopy 2, 8–10 (2003).
    Google Scholar
  10. Heymann, J.A. et al. J. Struct. Biol. 155, 63–73 (2006).
    Article Google Scholar
  11. Marko, M., Hsieh, C., MoberlyChan, W., Mannella, C.A. & Frank, J. J. Microsc. 222, 42–47 (2006).
    Article CAS Google Scholar
  12. Dubochet, J. et al. Q. Rev. Biophys. 21, 129–228 (1988).
    Article CAS Google Scholar
  13. Dubochet, J., McDowall, A.W., Menge, B., Schmid, E.N. & Lickfeld, K.G. J. Bact. 155, 381–390 (1983).
    CAS PubMed Google Scholar
  14. Matias, V.R.F., Al-Amoudi, A., Dubochet, J. & Beveridge, T.J. J. Bacteriol. 185, 6112–6118 (2003).
    Article CAS Google Scholar
  15. Zhang, P. et al. J. Microsc. 216, 76–83 (2004).
    Article CAS Google Scholar
  16. Marko, M. et al. Microsc. Microanal. 12 (Suppl. 2), 98–99 (2006).
    Article Google Scholar

Download references

Acknowledgements

We thank E. Currenti for providing the bacterial cells, and Wadsworth Center's EM Core for use of the high-pressure freezing facility. Supported by the US National Institutes of Health grant RR01219 (to J.F.), through the National Center for Research Resources Biomedical Research Technology Program.

Author information

Authors and Affiliations

  1. Resource for Visualization of Biological Complexity, Wadsworth Center, Empire State Plaza, Albany, 12201, New York, USA
    Michael Marko, Chyongere Hsieh, Joachim Frank & Carmen Mannella
  2. Center for Nanoscale Systems, Harvard University, 7 Divinity Ave., Cambridge, 02138, Massachusetts, USA
    Richard Schalek
  3. Howard Hughes Medical Institute, Wadsworth Center, Empire State Plaza, Albany, 12201, New York, USA
    Joachim Frank

Authors

  1. Michael Marko
    You can also search for this author inPubMed Google Scholar
  2. Chyongere Hsieh
    You can also search for this author inPubMed Google Scholar
  3. Richard Schalek
    You can also search for this author inPubMed Google Scholar
  4. Joachim Frank
    You can also search for this author inPubMed Google Scholar
  5. Carmen Mannella
    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence toMichael Marko.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

About this article

Cite this article

Marko, M., Hsieh, C., Schalek, R. et al. Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy.Nat Methods 4, 215–217 (2007). https://doi.org/10.1038/nmeth1014

Download citation