A cryogenic, coincident fluorescence, electron and ion beam microscope
- Delft University of Technology, Netherlands
- California Institute of Technology, United States
- Delmic B.V., Netherlands
- Leiden University Medical Center, Netherlands
- Max Planck Institute of Biochemistry, Germany
- Max Planck Institute of Molecular Physiology, Germany
- 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
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Data underlying the publication: A cryogenic, coincident fluorescence, electron and ion beam microscope4TU.ResearchData, doi:10.4121/20787274.
Article and author information
Author details
Ernest B van der Wee
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
- Suzanne R Pfeffer, Stanford University, United States
Version history
- Received: August 23, 2022
- Preprint posted: September 3, 2022 (view preprint)
- Accepted: October 25, 2022
- Accepted Manuscript published: October 28, 2022 (version 1)
- Version of Record published: December 1, 2022 (version 2)
- 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.
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A cryogenic, coincident fluorescence, electron and ion beam microscope
eLife 11:e82891.
https://doi.org/10.7554/eLife.82891
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