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  Cryo-FIB-SEM as a promising tool for localizing proteins in 3D

Spehner, D., Steyer, A. M., Bertinetti, L., Orlov, I., Benoit, L., Pernet-Gallay, K., et al. (2020). Cryo-FIB-SEM as a promising tool for localizing proteins in 3D. Journal of Structural Biology, 211(1): 107528. doi:10.1016/j.jsb.2020.107528.

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Item Permalink: http://hdl.handle.net/21.11116/0000-0006-5FB5-4 Version Permalink: http://hdl.handle.net/21.11116/0000-0006-9AF3-A
Genre: Journal Article

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 Creators:
Spehner, Daniele, Author
Steyer, Anna M., Author
Bertinetti, Luca1, Author              
Orlov, Igor, Author
Benoit, Lucas, Author
Pernet-Gallay, Karin, Author
Schertel, Andreas, Author
Schultz, Patrick, Author
Affiliations:
1Luca Bertinetti, Biomaterialien, Max Planck Institute of Colloids and Interfaces, Max Planck Society, ou_2379691              

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Free keywords: 3-D Cryo-FIB-SEM, Immunolabeling, Image processing and machine learning, 3D reconstruction and modelling
 Abstract: Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) is an invaluable tool to visualize the 3D architecture of cell constituents and map cell networks. Recently, amorphous ice embedding techniques have been associated with FIB-SEM to ensure that the biological material remains as close as possible to its native state. Here we have vitrified human HeLa cells and directly imaged them by cryo-FIB-SEM with the secondary electron InLens detector at cryogenic temperature and without any staining. Image stacks were aligned and processed by denoising, removal of ion beam milling artefacts and local charge imbalance. Images were assembled into a 3D volume and the major cell constituents were modelled. The data illustrate the power of the workflow to provide a detailed view of the internal architecture of the fully hydrated, close-to-native, entire HeLa cell. In addition, we have studied the feasibility of combining cryo-FIB-SEM imaging with live-cell protein detection. We demonstrate that internalized gold particles can be visualized by detecting back scattered primary electrons at low kV while simultaneously acquiring signals from the secondary electron detector to image major cell features. Furthermore, gold-conjugated antibodies directed against RNA polymerase II could be observed in the endo-lysosomal pathway while labelling of the enzyme in the nucleus was not detected, a shortcoming likely due to the inadequacy between the size of the gold particles and the voxel size. With further refinements, this method promises to have a variety of applications where the goal is to localize cellular antigens while visualizing the entire native cell in three dimensions.

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Language(s): eng - English
 Dates: 2020-05-062020
 Publication Status: Published in print
 Pages: -
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 Table of Contents: -
 Rev. Type: -
 Identifiers: DOI: 10.1016/j.jsb.2020.107528
 Degree: -

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Title: Journal of Structural Biology
  Abbreviation : J. Struct. Biol.
Source Genre: Journal
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Publ. Info: San Diego, CA : Elsevier
Pages: - Volume / Issue: 211 (1) Sequence Number: 107528 Start / End Page: - Identifier: ISSN: 1047-8477