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Bio Protoc. 2015 Sep 5;5(17). pii: e1575.

Cryo-focused Ion Beam Sample Preparation for Imaging Vitreous Cells by Cryo-electron Tomography.

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1
Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.

Abstract

Cryo-electron tomography (CET) is a well-established technique for imaging cellular and molecular structures at sub-nanometer resolution. As the method is limited to samples that are thinner than 500 nm, suitable sample preparation is required to attain CET data from larger cell volumes. Recently, cryo-focused ion beam (cryo-FIB) milling of plunge-frozen biological material has been shown to reproducibly yield large, homogeneously thin, distortion-free vitreous cross-sections for state-of-the-art CET. All eukaryotic and prokaryotic cells that can be plunge-frozen can be thinned with the cryo-FIB technique. Together with advances in low-dose microscopy, this has shifted the frontiers of in situ structural biology. In this protocol we describe the typical steps of the cryo-FIB technique, starting with fully grown cell cultures. Three recently investigated biological samples are given as examples.

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