A protocol for cryogenic volumetric imaging using serial plasma FIB/SEM.
Autor: | Dumoux M; Structural Biology, The Rosalind Franklin Institute, Harwell Science & Innovation Campus, Didcot, United Kingdom., Smith JLR; Structural Biology, The Rosalind Franklin Institute, Harwell Science & Innovation Campus, Didcot, United Kingdom; Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, United Kingdom., Glen T; Structural Biology, The Rosalind Franklin Institute, Harwell Science & Innovation Campus, Didcot, United Kingdom., Grange M; Structural Biology, The Rosalind Franklin Institute, Harwell Science & Innovation Campus, Didcot, United Kingdom; Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, United Kingdom., Darrow MC; Artificial Intelligence and Informatics, The Rosalind Franklin Institute, Harwell Science & Innovation Campus, Didcot, United Kingdom; SPT Labtech Ltd, Melbourn Science Park, Melbourn, United Kingdom. Electronic address: michele.darrow@rfi.ac.uk., Naismith JH; Structural Biology, The Rosalind Franklin Institute, Harwell Science & Innovation Campus, Didcot, United Kingdom; Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, United Kingdom. |
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Jazyk: | angličtina |
Zdroj: | Methods in cell biology [Methods Cell Biol] 2023; Vol. 177, pp. 327-358. Date of Electronic Publication: 2023 Mar 09. |
DOI: | 10.1016/bs.mcb.2023.01.015 |
Abstrakt: | Cryogenic volumetric imaging using serial plasma focused ion beam scanning electron microscopy (serial pFIB/SEM) is a new and exciting correlative volume electron microscopy (vEM) technique. It enables visualization of un-stained, cryogenically immobilized cells and tissues with ∼20-50nm resolution and a field of view of ∼10-30μm resulting in near-native state imaging and the possibility of microscale, mesoscale and nanoscale correlative imaging. We have written a detailed protocol for optimization of FIB and SEM parameters to reduce imaging artefacts and enable downstream computational processing and analysis. While our experience is based on use of a single system, the protocol has been written to be as hardware and software agnostic as possible, with a focus on the purpose of each step rather than a fully procedural description to provide a useful resource regardless of the system/software in use. Competing Interests: Conflict of interest MCD is an employee of SPT Labtech Ltd. The remaining authors declare no conflicts of interest. (Copyright © 2023 Elsevier Inc. All rights reserved.) |
Databáze: | MEDLINE |
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