CaltechAUTHORS
Published October 28, 2022 | Version Published
Journal Article Open

A cryogenic, coincident fluorescence, electron, and ion beam microscope

Creators

  • 1. ROR icon Delft University of Technology
  • 2. ROR icon California Institute of Technology
  • 3. ROR icon Brigham Young University
  • 4. ROR icon Leiden University Medical Center
  • 5. ROR icon Max Planck Institute of Biochemistry
  • 6. ROR icon Max Planck Institute of Molecular Physiology
  • 7. ROR icon University of Queensland

Abstract

Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre 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 three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope 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.

Additional Information

© 2022, Boltje et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Drosophila flight muscle myofibrils were kindly provided by EH Chan and F Schnorrer (Institut de Biologie du Développement de Marseille), and zebrafish myofibrils by Y Hinits and M Gautel (King's College London). We express our gratitude to Fulvio Reggiori (University of Groningen, Netherlands) for providing the HeLa cells and are thankful to Mingjun Xu for help during sample preparation. We thank Andries Effting (Delmic BV) for helpful discussions, and we would like to thank Ryan Lane (TU Delft) for his contribution in various Python developments. The majority of the 3D CAD design was done by Thomas van der Heijden (Delmic BV), for which we are grateful. This work was financially supported by NWO-TTW project no. 17152 to JPH, NIH grant RO1 AI127401 to GJJ, European SME2 grant no. 879673 to Delmic BV, Eurostars grant no. E13008 to SH & SR, and ERC grant ERC-StG-852880 to AJJ. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

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Additional details

Identifiers

PMCID
PMC9714966
Eprint ID
120345
Resolver ID
CaltechAUTHORS:20230322-367800000.31

Related works

Describes
https://resolver.caltech.edu/CaltechAUTHORS:20230322-367782000.30 (URL)
10.4121/20787274 (DOI)

Funding

Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)
17152
NIH
RO1 AI127401
European Research Council (ERC)
879673
Eurostars
E13008
European Research Council (ERC)
852880

Dates

Created
2023-03-26
Created from EPrint's datestamp field
Updated
2023-03-26
Created from EPrint's last_modified field

Caltech Custom Metadata

Caltech groups
Division of Biology and Biological Engineering (BBE)