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

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...

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Autores principales: Boltje, Daan B, Hoogenboom, Jacob P, Jakobi, Arjen J, Jensen, Grant J, Jonker, Caspar TH, Kaag, Max J, Koster, Abraham J, Last, Mart GF, de Agrela Pinto, Cecilia, Plitzko, Jürgen M, Raunser, Stefan, Tacke, Sebastian, Wang, Zhexin, van der Wee, Ernest B, Wepf, Roger, den Hoedt, Sander
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9714966/
https://www.ncbi.nlm.nih.gov/pubmed/36305590
http://dx.doi.org/10.7554/eLife.82891
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author Boltje, Daan B
Hoogenboom, Jacob P
Jakobi, Arjen J
Jensen, Grant J
Jonker, Caspar TH
Kaag, Max J
Koster, Abraham J
Last, Mart GF
de Agrela Pinto, Cecilia
Plitzko, Jürgen M
Raunser, Stefan
Tacke, Sebastian
Wang, Zhexin
van der Wee, Ernest B
Wepf, Roger
den Hoedt, Sander
author_facet Boltje, Daan B
Hoogenboom, Jacob P
Jakobi, Arjen J
Jensen, Grant J
Jonker, Caspar TH
Kaag, Max J
Koster, Abraham J
Last, Mart GF
de Agrela Pinto, Cecilia
Plitzko, Jürgen M
Raunser, Stefan
Tacke, Sebastian
Wang, Zhexin
van der Wee, Ernest B
Wepf, Roger
den Hoedt, Sander
author_sort Boltje, Daan B
collection PubMed
description 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.
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spelling pubmed-97149662022-12-02 A cryogenic, coincident fluorescence, electron, and ion beam microscope Boltje, Daan B Hoogenboom, Jacob P Jakobi, Arjen J Jensen, Grant J Jonker, Caspar TH Kaag, Max J Koster, Abraham J Last, Mart GF de Agrela Pinto, Cecilia Plitzko, Jürgen M Raunser, Stefan Tacke, Sebastian Wang, Zhexin van der Wee, Ernest B Wepf, Roger den Hoedt, Sander eLife Structural Biology and Molecular Biophysics 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. eLife Sciences Publications, Ltd 2022-10-28 /pmc/articles/PMC9714966/ /pubmed/36305590 http://dx.doi.org/10.7554/eLife.82891 Text en © 2022, Boltje et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Structural Biology and Molecular Biophysics
Boltje, Daan B
Hoogenboom, Jacob P
Jakobi, Arjen J
Jensen, Grant J
Jonker, Caspar TH
Kaag, Max J
Koster, Abraham J
Last, Mart GF
de Agrela Pinto, Cecilia
Plitzko, Jürgen M
Raunser, Stefan
Tacke, Sebastian
Wang, Zhexin
van der Wee, Ernest B
Wepf, Roger
den Hoedt, Sander
A cryogenic, coincident fluorescence, electron, and ion beam microscope
title A cryogenic, coincident fluorescence, electron, and ion beam microscope
title_full A cryogenic, coincident fluorescence, electron, and ion beam microscope
title_fullStr A cryogenic, coincident fluorescence, electron, and ion beam microscope
title_full_unstemmed A cryogenic, coincident fluorescence, electron, and ion beam microscope
title_short A cryogenic, coincident fluorescence, electron, and ion beam microscope
title_sort cryogenic, coincident fluorescence, electron, and ion beam microscope
topic Structural Biology and Molecular Biophysics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9714966/
https://www.ncbi.nlm.nih.gov/pubmed/36305590
http://dx.doi.org/10.7554/eLife.82891
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