Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

Fabian U. Zwettler, Sebastian Reinhard, Davide Gambarotto, Toby D. M. Bell, Virginie Hamel (), Paul Guichard () and Markus Sauer ()
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Fabian U. Zwettler: University of Würzburg, Am Hubland
Sebastian Reinhard: University of Würzburg, Am Hubland
Davide Gambarotto: Sciences III, University of Geneva
Toby D. M. Bell: Monash University
Virginie Hamel: Sciences III, University of Geneva
Paul Guichard: Sciences III, University of Geneva
Markus Sauer: University of Würzburg, Am Hubland

Nature Communications, 2020, vol. 11, issue 1, 1-11

Abstract: Abstract Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

Date: 2020
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DOI: 10.1038/s41467-020-17086-8

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