1. Structural Biology and Molecular Biophysics

A cryogenic, coincident fluorescence, electron and ion beam microscope

  1. Daan B Boltje  Is a corresponding author
  2. Jacob P Hoogenboom  Is a corresponding author
  3. Arjen J Jakobi
  4. Grant J Jensen
  5. Caspar TH Jonker
  6. Max J Kaag
  7. Abraham J Koster
  8. Mart GF Last
  9. Cecilia de Agrela Pinto
  10. Jürgen M Plitzko
  11. Stefan Raunser
  12. Sebastian Tacke
  13. Zhexin Wang
  14. Ernest B van der Wee
  15. Roger Wepf
  16. Sander den Hoedt
  1. Delft University of Technology, Netherlands
  2. California Institute of Technology, United States
  3. Delmic B.V., Netherlands
  4. Leiden University Medical Center, Netherlands
  5. Max Planck Institute of Biochemistry, Germany
  6. Max Planck Institute of Molecular Physiology, Germany
  7. University of Queensland, Australia
https://doi.org/10.7554/eLife.82891
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Cite this article
  1. Daan B Boltje
  2. Jacob P Hoogenboom
  3. Arjen J Jakobi
  4. Grant J Jensen
  5. Caspar TH Jonker
  6. Max J Kaag
  7. Abraham J Koster
  8. Mart GF Last
  9. Cecilia de Agrela Pinto
  10. Jürgen M Plitzko
  11. Stefan Raunser
  12. Sebastian Tacke
  13. Zhexin Wang
  14. Ernest B van der Wee
  15. Roger Wepf
  16. Sander den Hoedt
(2022)
A cryogenic, coincident fluorescence, electron and ion beam microscope
eLife 11:e82891.
https://doi.org/10.7554/eLife.82891
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  3. Download .RIS

Abstract

Cryogenic electron tomography (cryo-ET) combined with sub-tomogram averaging, allows in-situ visualization and structure determination of macromolecular complexes at sub-nanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in-situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident 3-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing focused ion-beam scanning electron microscope (FIB-SEM). We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope (TEM) tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

Data availability

The data underlying the publication can be found at international data repository service 4TU.ResearchData, https://doi.org/10.4121/20787274

The following data sets were generated

Article and author information

Author details

  1. Daan B Boltje

    Delft University of Technology, Delft, Netherlands
    For correspondence
    boltje@delmic.com
    Competing interests
    Daan B Boltje, is an employee of Delmic B.V..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4881-4700

  2. Jacob P Hoogenboom

    Delft University of Technology, Delft, Netherlands
    For correspondence
    J.P.Hoogenboom@TUDelft.nl
    Competing interests
    Jacob P Hoogenboom, has a financial interest in Delmic B.V..

  3. Arjen J Jakobi

    Delft University of Technology, Delft, Netherlands
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7761-2027

  4. Grant J Jensen

    Biology and Bioengineering, California Institute of Technology, Pasadena, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1556-4864

  5. Caspar TH Jonker

    Delmic B.V., Delft, Netherlands
    Competing interests
    Caspar TH Jonker, was an employee of Delmic B.V..

  6. Max J Kaag

    Delft University of Technology, Delft, Netherlands
    Competing interests
    No competing interests declared.

  7. Abraham J Koster

    Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, Netherlands
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1717-2549

  8. Mart GF Last

    Delmic B.V., Delft, Netherlands
    Competing interests
    Mart GF Last, was an employee of Delmic B.V..

  9. Cecilia de Agrela Pinto

    Delft University of Technology, Delft, Netherlands
    Competing interests
    No competing interests declared.

  10. Jürgen M Plitzko

    Department Molecular Structural Biology, 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

  11. Stefan Raunser

    Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9373-3016

  12. Sebastian Tacke

    Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Competing interests
    No competing interests declared.

  13. Zhexin Wang

    Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4256-1143

  14. Ernest B van der Wee

    Delft University of Technology, Delft, Netherlands
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0139-4019

  15. Roger Wepf

    Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, Australia
    Competing interests
    No competing interests declared.

  16. Sander den Hoedt

    Delmic B.V., Delft, Netherlands
    Competing interests
    Sander den Hoedt, has a financial interest in Delmic B.V..

Funding

Nederlandse Organisatie voor Wetenschappelijk Onderzoek (TTW No 17152)

  • Jacob P Hoogenboom

National Institutes of Health (RO1 AI127401)

  • Grant J Jensen

European Commission (SME2 No 879673)

  • Sander den Hoedt

Eurostars (No E13008)

  • Stefan Raunser
  • Sander den Hoedt

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

Reviewing Editor

  1. Suzanne R Pfeffer, Stanford University, United States

Publication history

  1. Received: August 23, 2022
  2. Preprint posted: September 3, 2022 (view preprint)
  3. Accepted: October 25, 2022
  4. Accepted Manuscript published: October 28, 2022 (version 1)
  5. Version of Record published: December 1, 2022 (version 2)

Copyright

© 2022, Boltje 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. Daan B Boltje
  2. Jacob P Hoogenboom
  3. Arjen J Jakobi
  4. Grant J Jensen
  5. Caspar TH Jonker
  6. Max J Kaag
  7. Abraham J Koster
  8. Mart GF Last
  9. Cecilia de Agrela Pinto
  10. Jürgen M Plitzko
  11. Stefan Raunser
  12. Sebastian Tacke
  13. Zhexin Wang
  14. Ernest B van der Wee
  15. Roger Wepf
  16. Sander den Hoedt
(2022)
A cryogenic, coincident fluorescence, electron and ion beam microscope
eLife 11:e82891.
https://doi.org/10.7554/eLife.82891

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