Abstract

Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of sub-cellular structures on the mesoscale (10 nm to 10 µm). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents' hydration shells for in-situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining, and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM (cryo-pFIB/SEM). We evaluated the choice of plasma ion source and imaging regimes to produce high quality SEM images of a range of different biological samples. Using an automated workflow we produced three dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20 to 50 nm. Additionally, a tag-free localisation tool for regions of interest is needed to drive the application of in-situ structural biology towards tissue. The combination of serial FIB/SEM with plasma-based ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 µm) to produce lamellae for cryogenic electron tomography.

Data availability

Raw data, along with segmentation and associated TEM overviews (if relevant) are deposited on the EMPIAR data repository: EMPIAR-11414, EMPIAR-11415, EMPIAR-11416, EMPIAR-11417, EMPIAR-11418, EMPIAR-11419, EMPIAR-11420, EMPIAR-11421. Code can be found on the Rosalind Franklin Institute GitHub (https://github.com/rosalindfranklininstitute/) and the serialFIB GitHub (https://github.com/sklumpe/SerialFIB/).

The following data sets were generated

Article and author information

Author details

  1. Maud Dumoux

    Structural Biology, Rosalind Franklin Institute, Didcot, United Kingdom
    For correspondence
    maud.dumoux@rfi.ac.uk
    Competing interests
    No competing interests declared.
  2. Thomas Glen

    Structural Biology, Rosalind Franklin Institute, Didcot, United Kingdom
    Competing interests
    No competing interests declared.
  3. Jake LR Smith

    Structural Biology, Rosalind Franklin Institute, Didcot, United Kingdom
    Competing interests
    No competing interests declared.
  4. Elaine ML Ho

    Artificial Intelligence and Informatics, Rosalind Franklin Institute, Didcot, United Kingdom
    Competing interests
    No competing interests declared.
  5. Luis MA Perdigão

    Artificial Intelligence and Informatics, Rosalind Franklin Institute, Didcot, United Kingdom
    Competing interests
    No competing interests declared.
  6. Avery Pennington

    Diamond Light Source, Didcot, United Kingdom
    Competing interests
    No competing interests declared.
  7. Sven Klumpe

    Research Group Cryo-EM Technology, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    No competing interests declared.
  8. Neville BY Yee

    Artificial Intelligence and Informatics, Rosalind Franklin Institute, Didcot, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0349-3958
  9. David Andrew Farmer

    Diamond Light Source, Didcot, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5331-3551
  10. Pui YA Lai

    Diamond Light Source, Didcot, United Kingdom
    Competing interests
    No competing interests declared.
  11. William Bowles

    Structural Biology, Rosalind Franklin Institute, Didcot, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8115-4404
  12. Ron Kelley

    Materials and Structural Analysis Division, Thermo Fisher Scientific, Eindhoven, Netherlands
    Competing interests
    Ron Kelley, is an employee of ThermoFisher Scientific.
  13. Jürgen M Plitzko

    Research Group Cryo-EM Technology, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6402-8315
  14. Liang Wu

    Structural Biology, Rosalind Franklin Institute, Didcot, United Kingdom
    Competing interests
    No competing interests declared.
  15. Mark Basham

    Diamond Light Source, Didcot, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8438-1415
  16. Daniel K Clare

    Diamond Light Source, Didcot, United Kingdom
    Competing interests
    No competing interests declared.
  17. C Alistair Siebert

    eBIC, Diamond Light Source, Didcott, United Kingdom
    Competing interests
    No competing interests declared.
  18. Michele C Darrow

    Artificial Intelligence and Informatics, Rosalind Franklin Institute, Didcot, United Kingdom
    Competing interests
    Michele C Darrow, is an employee of SPT Labtech..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6259-1684
  19. James H Naismith

    Structural Biology, Rosalind Franklin Institute, Didcot, United Kingdom
    Competing interests
    No competing interests declared.
  20. Michael Grange

    Structural Biology, Rosalind Franklin Institute, Didcot, United Kingdom
    For correspondence
    michael.grange@rfi.ac.uk
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2580-2299

Funding

Wellcome Trust (220526/Z/20/Z)

  • James H Naismith

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

Reviewing Editor

  1. Giulia Zanetti, Birkbeck, University of London, United Kingdom

Ethics

Animal experimentation: All of the animals were handled according to approved, reviewed institutional animal care procedures. Mice were euthanised in a schedule 1 procedure via intraperitoneal injection of sodium pentobarbital followed by decapitation following licensed procedures approved by the Mary Lyon Centre and the Home Office UK. All operating procedures were designed to minimise any suffering for the animals involved in the study.

Version history

  1. Preprint posted: September 21, 2022 (view preprint)
  2. Received: September 21, 2022
  3. Accepted: February 20, 2023
  4. Accepted Manuscript published: February 21, 2023 (version 1)
  5. Version of Record published: March 8, 2023 (version 2)

Copyright

© 2023, Dumoux 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. Maud Dumoux
  2. Thomas Glen
  3. Jake LR Smith
  4. Elaine ML Ho
  5. Luis MA Perdigão
  6. Avery Pennington
  7. Sven Klumpe
  8. Neville BY Yee
  9. David Andrew Farmer
  10. Pui YA Lai
  11. William Bowles
  12. Ron Kelley
  13. Jürgen M Plitzko
  14. Liang Wu
  15. Mark Basham
  16. Daniel K Clare
  17. C Alistair Siebert
  18. Michele C Darrow
  19. James H Naismith
  20. Michael Grange
(2023)
Cryo-plasma FIB/SEM volume imaging of biological specimens
eLife 12:e83623.
https://doi.org/10.7554/eLife.83623

Share this article

https://doi.org/10.7554/eLife.83623

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