Fully automated, sequential focused ion beam milling for cryo-electron tomography

  1. Tobias Zachs
  2. Andreas Schertel
  3. João Medeiros
  4. Gregor L Weiss
  5. Jannik Hugener
  6. Joao Matos
  7. Martin Pilhofer  Is a corresponding author
  1. ETH Zürich, Switzerland
  2. 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

The following data sets were generated

Article and author information

Author details

  1. Tobias Zachs

    Institute of Molecular Biology and Biophysics, ETH Zürich, Zürich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  2. Andreas Schertel

    Zeiss Customer Center Europe, Carl Zeiss Microscopy GmbH, Oberkochen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. João Medeiros

    Department of Biology, ETH Zürich, Zürich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9075-548X
  4. Gregor L Weiss

    Department of Biology, ETH Zürich, Zürich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  5. Jannik Hugener

    Department of Biology, ETH Zürich, Zürich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  6. Joao Matos

    Institute of Biochemistry, ETH Zürich, Zürich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3754-3709
  7. Martin Pilhofer

    Department of Biology, ETH Zürich, Zürich, Switzerland
    For correspondence
    pilhofer@biol.ethz.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3649-3340

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

  1. Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom

Version history

  1. Received: September 28, 2019
  2. Accepted: March 7, 2020
  3. Accepted Manuscript published: March 9, 2020 (version 1)
  4. 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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  1. Tobias Zachs
  2. Andreas Schertel
  3. João Medeiros
  4. Gregor L Weiss
  5. Jannik Hugener
  6. Joao Matos
  7. Martin Pilhofer
(2020)
Fully automated, sequential focused ion beam milling for cryo-electron tomography
eLife 9:e52286.
https://doi.org/10.7554/eLife.52286

Share this article

https://doi.org/10.7554/eLife.52286

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