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

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

Version 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)
  6. Version of Record updated: April 20, 2023 (version 3)

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.

Metrics

  • 2,411
    views
  • 349
    downloads
  • 7
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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

Share this article

https://doi.org/10.7554/eLife.82891

Further reading

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Shun Kai Yang, Shintaroh Kubo ... Khanh Huy Bui
    Research Article

    Acetylation of α-tubulin at the lysine 40 residue (αK40) by αTAT1/MEC-17 acetyltransferase modulates microtubule properties and occurs in most eukaryotic cells. Previous literatures suggest that acetylated microtubules are more stable and damage resistant. αK40 acetylation is the only known microtubule luminal post-translational modification site. The luminal location suggests that the modification tunes the lateral interaction of protofilaments inside the microtubule. In this study, we examined the effect of tubulin acetylation on the doublet microtubule (DMT) in the cilia of Tetrahymena thermophila using a combination of cryo-electron microscopy, molecular dynamics, and mass spectrometry. We found that αK40 acetylation exerts a small-scale effect on the DMT structure and stability by influencing the lateral rotational angle. In addition, comparative mass spectrometry revealed a link between αK40 acetylation and phosphorylation in cilia.

    1. Structural Biology and Molecular Biophysics
    Sebastian Jojoa-Cruz, Adrienne E Dubin ... Andrew B Ward
    Research Advance

    The dimeric two-pore OSCA/TMEM63 family has recently been identified as mechanically activated ion channels. Previously, based on the unique features of the structure of OSCA1.2, we postulated the potential involvement of several structural elements in sensing membrane tension (Jojoa-Cruz et al., 2018). Interestingly, while OSCA1, 2, and 3 clades are activated by membrane stretch in cell-attached patches (i.e. they are stretch-activated channels), they differ in their ability to transduce membrane deformation induced by a blunt probe (poking). Here, in an effort to understand the domains contributing to mechanical signal transduction, we used cryo-electron microscopy to solve the structure of Arabidopsis thaliana (At) OSCA3.1, which, unlike AtOSCA1.2, only produced stretch- but not poke-activated currents in our initial characterization (Murthy et al., 2018). Mutagenesis and electrophysiological assessment of conserved and divergent putative mechanosensitive features of OSCA1.2 reveal a selective disruption of the macroscopic currents elicited by poking without considerable effects on stretch-activated currents (SAC). Our results support the involvement of the amphipathic helix and lipid-interacting residues in the membrane fenestration in the response to poking. Our findings position these two structural elements as potential sources of functional diversity within the family.