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  Fluorescence-Based Detection of Membrane Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy

Metskas, L. A., & Briggs, J. A. G. (2019). Fluorescence-Based Detection of Membrane Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy. Microscopy and Microanalysis, 25(4), 942-949. doi:10.1017/s1431927619000606.

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Metskas, L. A., Author
Briggs, John A. G.1, 2, Author           
Affiliations:
1European Molecular Biology Laboratory, External Organizations, ou_3346677              
2MRC Laboratory of Molecular Biology, External Organizations, Cambridge, GB, ou_3346673              

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Free keywords: CLEM cryo-EM FRET membrane fusion resonant energy transfer superresolution fluorescence electron light tomography image Materials Science Microscopy
 Abstract: Correlated light and electron microscopy (CLEM) has become a popular technique for combining the protein-specific labeling of fluorescence with electron microscopy, both at room and cryogenic temperatures. Fluorescence applications at cryo-temperatures have typically been limited to localization of tagged protein oligomers due to known issues of extended triplet state duration, spectral shifts, and reduced photon capture through cryo-CLEM objectives. Here, we consider fluorophore characteristics and behaviors that could enable more extended applications. We describe how dialkylcarbocanine DiD, and its autoquenching by resonant energy transfer (RET), can be used to distinguish the fusion state of a lipid bilayer at cryo-temperatures. By adapting an established fusion assay to work under cryo-CLEM conditions, we identified areas of fusion between influenza virus-like particles and fluorescently labeled lipid vesicles on a cryo-EM grid. This result demonstrates that cryo-CLEM can be used to localize functions in addition to tagged proteins, and that fluorescence autoquenching by RET can be incorporated successfully into cryo-CLEM approaches. In the case of membrane fusion applications, this method provides both an orthogonal confirmation of functional state independent of the morphological description from cryo-EM and a way to bridge room-temperature kinetic assays and the cryo-EM images.

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Language(s): eng - English
 Dates: 2019
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Identifiers: Other: WOS:000485052600012
DOI: 10.1017/s1431927619000606
ISSN: 1431-9276
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Title: Microscopy and Microanalysis
  Alternative Title : Microsc. microanal.
Source Genre: Journal
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Publ. Info: -
Pages: - Volume / Issue: 25 (4) Sequence Number: - Start / End Page: 942 - 949 Identifier: -