Imaging Microtubules in vitro at High Resolution While Preserving Their Structure
Autor: | Yasmina Saoudi, Camille Cuveillier, Isabelle Arnal, Christian Delphin |
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Rok vydání: | 2021 |
Předmět: |
Total internal reflection fluorescence microscope
Microscope Chemistry Strategy and Management Mechanical Engineering Confocal Resolution (electron density) Metals and Alloys Context (language use) Industrial and Manufacturing Engineering law.invention Microtubule law Microscopy Biophysics Electron microscope |
Zdroj: | BIO-PROTOCOL. 11 |
ISSN: | 2331-8325 |
Popis: | Microtubules (MT) are the most rigid component of the cytoskeleton. Nevertheless, they often appear highly curved in the cellular context and the mechanisms governing their overall shape are poorly understood. Currently, in vitro microtubule analysis relies primarily on electron microscopy for its high resolution and Total Internal Reflection Fluorescence (TIRF) microscopy for its ability to image live fluorescently-labelled microtubules and associated proteins. For three-dimensional analyses of microtubules with micrometer curvatures, we have developed an assay in which MTs are polymerized in vitro from MT seeds adhered to a glass slide in a manner similar to conventional TIRF microscopy protocols. Free fluorescent molecules are removed and the MTs are fixed by perfusion. The MTs can then be observed using a confocal microscope with an Airyscan module for higher resolution. This protocol allows the imaging of microtubules that have retained their original three-dimensional shape and is compatible with high-resolution immunofluorescence detection. |
Databáze: | OpenAIRE |
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