Robust workflow and instrumentation for cryo-focused ion beam milling of samples for electron cryotomography - PubMed (original) (raw)

Robust workflow and instrumentation for cryo-focused ion beam milling of samples for electron cryotomography

João M Medeiros et al. Ultramicroscopy. 2018 Jul.

Abstract

Electron cryotomography is able to visualize macromolecular complexes in their cellular context, in a frozen-hydrated state, and in three dimensions. The method, however, is limited to relatively thin samples. Cryo-focused ion beam (FIB) milling is emerging as a powerful technique for sample thinning prior to cryotomography imaging. Previous cryo-FIB milling reports utilized custom-built non-standard equipment. Here we present a workflow and the required commercially available instrumentation to either implement the method de novo, or as an upgrade of pre-existing dual beam milling instruments. We introduce two alternative protocols and the respective sample holders for milling. The "bare grid holder" allows for milling on plain grids, having the advantage of enabling relatively shallow milling angles for wedge geometries. The "Autogrid holder" is designed for milling grids clamped into a mechanical support ring (Autogrid), resulting in increased stability for lamella geometries. We applied the workflow to prepare samples and record high-quality tomograms of diverse model organisms, including infected and uninfected HeLa cells, amoebae, yeast, multicellular cyanobacteria, Pseudomonas aeruginosa and Escherichia coli cells. The workflow will contribute to the dissemination of electron cryotomography of cryo-FIB milled samples in the biological sciences.

Keywords: Cryo-electron microscopy; Cryo-focused ion beam milling; Electron cryotomography; In situ imaging; Sample holder.

Copyright © 2018 Elsevier B.V. All rights reserved.

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