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J Struct Biol. 2019 Dec 1;208(3). pii: S1047-8477(19)30200-X. doi: 10.1016/j.jsb.2019.09.006. Epub 2019 Sep 16.

Mind the gap: Micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae.

Author information

1
Department of Cell and Chemical Biology, Section Electron Microscopy, Leiden University Medical Center, Leiden 2333 ZC, The Netherlands.
2
Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA 94143, United States.
3
Department of Medical Microbiology, Molecular Virology Laboratory, Leiden University Medical Center, Leiden 2333 ZA, The Netherlands.
4
Department of Cell and Chemical Biology, Section Electron Microscopy, Leiden University Medical Center, Leiden 2333 ZC, The Netherlands. Electronic address: m.barcena@lumc.nl.

Abstract

Cryo-focused ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-lamella workflow is a low-throughput technique and can easily be hindered by technical issues like the bending of the lamellae during the final cryo-FIB-milling steps. The severity of lamella bending seems to correlate with crinkling of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin lamella, leading to its bending and breakage. To protect the lamellae from such forces, we milled "micro-expansion joints" alongside the lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of micro-expansion joints drastically decreases bending of lamellae milled from eukaryotic cells grown and frozen on EM grids. Furthermore, we show that this adaptation does not create additional instabilities that could impede subsequent parts of the cryo-lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them a straightforward solution against cryo-lamella bending to increase the throughput of in situ structural biology studies.

KEYWORDS:

Cellular cryotomography; Cryo-FIB-milling; Cryo-electron tomography; Cryo-lamella; Frozen-hydrated cells; Lamella bending

PMID:
31536774
DOI:
10.1016/j.jsb.2019.09.006
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