A cryogenic, coincident fluorescence, electron and ion beam microscope

Daan B. Boltje*, Jacob P. Hoogenboom, Arjen J. Jakobi, Grant J. Jensen, Caspar T.H. Jonker, Max J. Kaag, Cecilia de Agrela Pinto, Ernest B. van der Wee, Sander Den Hoedt, More Authors

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

3 Citations (Scopus)
15 Downloads (Pure)

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.

Original languageEnglish
Article numbere82891
Number of pages19
JournaleLife
Volume11
DOIs
Publication statusPublished - 2022

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