Correlated Cryo Super-Resolution Light and Cryo-Electron Microscopy on Mammalian Cells Expressing the Fluorescent Protein rsEGFP2
| Autor: | Maarten W. Tuijtel, Thomas H. Sharp, Abraham J. Koster, Frank G. A. Faas |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
chemistry.chemical_classification
0303 health sciences Materials science Super-resolution microscopy Cryo-electron microscopy Biomolecule 02 engineering and technology General Chemistry 021001 nanoscience & nanotechnology Superresolution Fluorescence cryoEM 03 medical and health sciences Biological specimen chemistry super-resolution microscopy Microscopy Fluorescent protein General Materials Science correlative light and electron microscopy 0210 nano-technology cryo-fluorescence microscopy 030304 developmental biology Biomedical engineering |
| Zdroj: | Small Methods. WILEY-V C H VERLAG GMBH Small Methods |
| Popis: | Super-resolution light microscopy (SRM) enables imaging of biomolecules within cells with nanometer precision. Cryo-fixation by vitrification offers optimal structure preservation of biological specimens and permits sequential cryo electron microscopy (cryoEM) on the same sample, but is rarely used for SRM due to various technical challenges and the lack of fluorophores developed for vitrified conditions. Here, a protocol to perform correlated cryoSRM and cryoEM on intact mammalian cells using fluorescent proteins and commercially available equipment is described. After cell culture and sample preparation by plunge-freezing, cryoSRM is performed using the reversibly photoswitchable fluorescent protein rsEGFP2. Next, a super-resolved image is reconstructed to guide cryoEM imaging to the feature of interest. Finally, the cryoSRM and cryoEM images are correlated to combine information from both imaging modalities. Using this protocol, a localization precision of 30 nm for cryoSRM is routinely achieved. No impediments to successive cryoEM imaging are detected, and the protocol is compatible with a variety of cryoEM techniques. When the optical set-up and analysis pipeline is established, the total duration of the protocol for experienced cryoEM users is 3 days, not including cell culture. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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