Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells
| Autor: | Kamal L Nahas, Mohamed A. Koronfel, Ian M. Dobbie, Nina Vyas, Maria Harkiolaki, Nina Perry, Thomas M. Fish, Archana Jadhav, Chidinma A. Okolo, Eva Pereiro, Johannes Groen, Ilias Kounatidis |
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| Rok vydání: | 2021 |
| Předmět: |
Microscope
Materials science Cells General Chemical Engineering Cytological Techniques General Biochemistry Genetics and Molecular Biology law.invention 03 medical and health sciences Imaging Three-Dimensional Tubulin law Microscopy Fluorescence microscope Humans Lighting Fluorescent Dyes 030304 developmental biology Cryopreservation 0303 health sciences General Immunology and Microbiology Super-resolution microscopy Data Collection General Neuroscience 030302 biochemistry & molecular biology Resolution (electron density) Synchrotron Microscopy Fluorescence Tomography Electron microscope Biomedical engineering |
| Zdroj: | Journal of Visualized Experiments |
| ISSN: | 1940-087X |
| DOI: | 10.3791/62274 |
| Popis: | Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than conventional fluorescence microscopy. This super-resolution (SR) technique enables visualization of molecular processes in whole cells and has the potential to be used in conjunction with electron microscopy and X-ray tomography to correlate structural and functional information. A SIM microscope for cryogenically preserved samples (cryoSIM) has recently been commissioned at the correlative cryo-imaging beamline B24 at the UK synchrotron. It was designed specifically for 3D imaging of biological samples at cryogenic temperatures in a manner compatible with subsequent imaging of the same samples by X-ray microscopy methods such as cryo-soft X-ray tomography. This video article provides detailed methods and protocols for successful imaging using the cryoSIM. In addition to instructions on the operation of the cryoSIM microscope, recommendations have been included regarding the choice of samples, fluorophores, and parameter settings. The protocol is demonstrated in U2OS cell samples whose mitochondria and tubulin have been fluorescently labelled. |
| Databáze: | OpenAIRE |
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