| Autor: |
Reinhard Rachel, Ralph Witzgall, Jantina Fokkema, Uwe de Vries, Kerstin N. Schmidt, Olga Maier, Yulia Zaytseva, Hans C. Gerritsen, Korbinian Buerger |
| Rok vydání: |
2021 |
| Předmět: |
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| Zdroj: |
Methods in Cell Biology ISBN: 9780128220580 |
| Popis: |
The application of both fluorescence and electron microscopy results in a powerful combination of imaging modalities called "correlative light and electron microscopy" (CLEM). Whereas conventional transmission electron microscopy (TEM) tomography is only able to image sections up to a thickness of ~300nm, scanning transmission electron microscopy (STEM) tomography at 200kV allows the analysis of sections up to a thickness of 900nm in three dimensions. In the current study we have successfully integrated STEM tomography into CLEM as demonstrated for human retinal pigment epithelial 1 (RPE1) cells expressing various fluorescent fusion proteins which were high-pressure frozen and then embedded in Lowicryl HM20. Fluorescently labeled gold nanoparticles were applied onto resin sections and imaged by fluorescence and electron microscopy. STEM tomograms were recorded at regions of interest, and overlays were generated using the eC-CLEM software package. Through the nuclear staining of living cells, the use of fluorescently labeled gold fiducials for the generation of overlays, and the integration of STEM tomography we have markedly extended the application of the Kukulski protocol (Kukulski et al., 2011, 2012). Various fluorescently tagged proteins localizing to different cellular organelles could be assigned to their ultrastructural compartments. By combining STEM tomography with on-section CLEM, fluorescently tagged proteins can be localized in three-dimensional ultrastructural environments with a volume of at least 2.7×2.7×0.5μm. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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