Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)
| Autor: | Paul Guichard, Virginie Hamel, Sebastian Reinhard, Fabian U. Zwettler, Toby D. M. Bell, Davide Gambarotto, Markus Sauer |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Fluorescence-lifetime imaging microscopy
Microscope Normal Distribution General Physics and Astronomy 02 engineering and technology Microtubules law.invention Electrolytes Epitopes chemistry.chemical_compound 0302 clinical medicine law Chlorocebus aethiops Microscopy Denaturation (biochemistry) Super-resolution microscopy lcsh:Science Centrioles 0303 health sciences Multidisciplinary Hydrogels 021001 nanoscience & nanotechnology Single Molecule Imaging COS Cells Self-healing hydrogels 0210 nano-technology Fluorophore Materials science Photochemistry Science Buffers Article General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Imaging Three-Dimensional Single-molecule biophysics Animals Computer Simulation Fluorescent Dyes 030304 developmental biology General Chemistry Microscopy Fluorescence chemistry Ultrastructure Biophysics lcsh:Q Biophotonics Electron microscope Chlamydomonas reinhardtii 030217 neurology & neurosurgery |
| Zdroj: | Nature Communications Nature Communications, Vol 11, Iss 1, Pp 1-11 (2020) |
| ISSN: | 2041-1723 |
| DOI: | 10.1038/s41467-020-17086-8 |
| Popis: | 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. Previous attempts to combine expansion microscopy (ExM) and single molecule localisation microscopy (SMLM) have proved challenging. Here the authors show that post-labelling Ex-SMLM improves labelling efficiency, reduces linkage error, and preserves ultrastructural details. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|