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Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy

Nature Methods volume 4, pages 215217 (2007) | Download Citation

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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.

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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

Affiliations

  1. Resource for Visualization of Biological Complexity, Wadsworth Center, Empire State Plaza, Albany, New York 12201, USA.

    • Michael Marko
    • , Chyongere Hsieh
    • , Joachim Frank
    •  & Carmen Mannella
  2. Center for Nanoscale Systems, Harvard University, 7 Divinity Ave., Cambridge, Massachusetts 02138, USA.

    • Richard Schalek
  3. Howard Hughes Medical Institute, Wadsworth Center, Empire State Plaza, Albany, New York 12201, USA.

    • Joachim Frank

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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Michael Marko.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Tomographic reconstruction of FIB-milled frozen-hydrated bacterium.

  2. 2.

    Supplementary Fig. 2

    Cryo-EM of E. coli cells prepared by cryo-ultramicrotomy.

  3. 3.

    Supplementary Methods

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DOI

https://doi.org/10.1038/nmeth1014

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