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

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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Figure 1: FIB milling of frozen-hydrated E. coli.
Figure 2: Cryo-electron tomograms of E. coli cells.

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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.

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Correspondence to Michael Marko.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Tomographic reconstruction of FIB-milled frozen-hydrated bacterium. (PDF 497 kb)

Supplementary Fig. 2

Cryo-EM of E. coli cells prepared by cryo-ultramicrotomy. (PDF 165 kb)

Supplementary Methods (PDF 68 kb)

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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

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