Fully automated, sequential focused ion beam milling for cryo-electron tomography
- ETH Zürich, Switzerland
- Carl Zeiss Microscopy GmbH, Germany
Abstract
Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.
Data availability
The tomography data are deposited at EMDB/EMPIAR.EMPIAR-10376, EMD-10707, EMD-10708, EMD-10710
Article and author information
Author details
Funding
Swiss National Science Foundation (#31003A_179255)
- Martin Pilhofer
European Research Council (#679209)
- Martin Pilhofer
Nomis Foundation (nd)
- Martin Pilhofer
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom
Publication history
- Received: September 28, 2019
- Accepted: March 7, 2020
- Accepted Manuscript published: March 9, 2020 (version 1)
- Version of Record published: March 19, 2020 (version 2)
Copyright
© 2020, Zachs et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Fully automated, sequential focused ion beam milling for cryo-electron tomography
eLife 9:e52286.
https://doi.org/10.7554/eLife.52286
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