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J Struct Biol. 2012 Nov;180(2):318-26. doi: 10.1016/j.jsb.2012.07.003. Epub 2012 Jul 14.

3D structure determination of native mammalian cells using cryo-FIB and cryo-electron tomography.

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1
Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Ave, Pittsburgh, PA 15260, USA.

Abstract

Cryo-electron tomography (cryo-ET) has enabled high resolution three-dimensional(3D) structural analysis of virus and host cell interactions and many cell signaling events; these studies, however, have largely been limited to very thin, peripheral regions of eukaryotic cells or to small prokaryotic cells. Recent efforts to make thin, vitreous sections using cryo-ultramicrotomy have been successful, however,this method is technically very challenging and with many artifacts. Here, we report a simple and robust method for creating in situ, frozen-hydrated cell lamellas using a focused ion beam at cryogenic temperature (cryo-FIB), allowing access to any interior cellular regions of interest. We demonstrate the utility of cryo-FIB with high resolution 3D cellular structures from both bacterial cells and large mammalian cells. The method will not only facilitate high-throughput 3D structural analysis of biological specimens, but is also broadly applicable to sample preparation of thin films and surface materials without the need for FIB "lift-out".

PMID:
22796867
PMCID:
PMC3483467
DOI:
10.1016/j.jsb.2012.07.003
[Indexed for MEDLINE]
Free PMC Article
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