A modular platform for automated cryo-FIB workflows

  1. Sven Klumpe
  2. Herman K H Fung
  3. Sara K Goetz
  4. Ievgeniia Zagoriy
  5. Bernhard Hampoelz
  6. Xiaojie Zhang
  7. Philipp S Erdmann
  8. Janina Baumbach
  9. Christoph W Müller
  10. Martin Beck
  11. Jürgen M Plitzko  Is a corresponding author
  12. Julia Mahamid  Is a corresponding author
  1. Max Planck Institute of Biochemistry, Germany
  2. European Molecular Biology Laboratory, Germany
  3. Max Planck Institute of Biophysics, Germany

Abstract

Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with 3-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.

Data availability

All code developed in this work is available on GitHub. SerialFIB and a written tutorial can be obtained on: https://github.com/sklumpe/SerialFIB. A comprehensive video tutorial session is uploaded to YouTube: https://www.youtube.com/watch?v=QR7ngJ0apBk. The documentation is available on GitHub (https://github.com/sklumpe/SerialFIB/blob/main/documentation/SFIB.pdf). A Python script for post-processing of cryo-FIB-SEM volume imaging data as described in the method section is available in the same repository. The Python 3-ported 3DCT is available on: https://github.com/hermankhfung/3dct. New 3DCT functions described in this work, scripts for cryo-FLM virtual slice series creation, 3D point-based registration and transformation of cryo-FLM data with respect to cryo-FIB-SEM data, and bUnwarpJ-based analysis of FIB images before and after milling are available on: https://github.com/hermankhfung/tools3dct. All tomograms and subtomogram averages depicted in the figures are deposited to EMDB under accession codes EMD-13832, EMD-13833, EMD-13836, EMD-13837, EMD-13838, EMD-13834, EMD-13835. FIB-SEM volume imaging data for the Sum159 cells and Chlamydomonas reinhardtii are deposited in EMPIAR under accession codes EMPIAR-10847, EMPIAR-10870. FIB, SEM, TEM and fluorescence images used for 3D correlation are available on the BioImage Archive (S-BSST730, S-BSST729). Numerical data for lamella widths and thicknesses presented in Figure 2-figure supplement 4 are tabulated in Figure 2-figure supplement 4-Source Data 1 and 2.

Article and author information

Author details

  1. Sven Klumpe

    Department Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    No competing interests declared.
  2. Herman K H Fung

    Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    Competing interests
    No competing interests declared.
  3. Sara K Goetz

    Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    Competing interests
    No competing interests declared.
  4. Ievgeniia Zagoriy

    Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    Competing interests
    No competing interests declared.
  5. Bernhard Hampoelz

    Department Molecular Sociology, Max Planck Institute of Biophysics, Frankfurt, Germany
    Competing interests
    No competing interests declared.
  6. Xiaojie Zhang

    Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    Competing interests
    No competing interests declared.
  7. Philipp S Erdmann

    Department Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    No competing interests declared.
  8. Janina Baumbach

    Department Molecular Sociology, Max Planck Institute of Biophysics, Frankfurt, Germany
    Competing interests
    No competing interests declared.
  9. Christoph W Müller

    Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2176-8337
  10. Martin Beck

    Department Molecular Sociology, Max Planck Institute of Biophysics, Frankfurt, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7397-1321
  11. Jürgen M Plitzko

    Department Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany
    For correspondence
    plitzko@biochem.mpg.de
    Competing interests
    Jürgen M Plitzko, holds a position on the advisory board of Thermo Fisher Scientific..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6402-8315
  12. Julia Mahamid

    Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    For correspondence
    julia.mahamid@embl.de
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6968-041X

Funding

European research council (760067)

  • Julia Mahamid

European research council (871037)

  • Julia Mahamid

EMBL Interdisciplinary Postdoctoral Program under Marie Curie Actions COFUND (664726)

  • Herman K H Fung

Max Planck Institute for Biochrmistry

  • Sven Klumpe

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Axel T Brunger, Stanford University School of Medicine, Howard Hughes Medical Institute, United States

Publication history

  1. Received: May 18, 2021
  2. Preprint posted: May 20, 2021 (view preprint)
  3. Accepted: December 23, 2021
  4. Accepted Manuscript published: December 24, 2021 (version 1)
  5. Version of Record published: January 17, 2022 (version 2)

Copyright

© 2021, Klumpe 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. Sven Klumpe
  2. Herman K H Fung
  3. Sara K Goetz
  4. Ievgeniia Zagoriy
  5. Bernhard Hampoelz
  6. Xiaojie Zhang
  7. Philipp S Erdmann
  8. Janina Baumbach
  9. Christoph W Müller
  10. Martin Beck
  11. Jürgen M Plitzko
  12. Julia Mahamid
(2021)
A modular platform for automated cryo-FIB workflows
eLife 10:e70506.
https://doi.org/10.7554/eLife.70506

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