Single Molecule Fluorescence Microscopy on Planar Supported Bilayers
| Autor: | Johannes B. Huppa, Markus Axmann, Gerhard J. Schütz |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Materials science
General Chemical Engineering Lipid Bilayers Total internal reflection microscopy Laser Nanotechnology Bioengineering General Biochemistry Genetics and Molecular Biology Exocytosis Planar Glass-Supported Lipid Bilayer 03 medical and health sciences 0302 clinical medicine Microscopy Small/Large Unilamellar Vesicles Lipid bilayer Total Internal Reflection Microscopy 030304 developmental biology 0303 health sciences Total internal reflection Total internal reflection fluorescence microscope General Immunology and Microbiology General Neuroscience Resolution (electron density) Cell Membrane Proteins Single-molecule experiment Single Molecule Imaging Molecular Imaging Microscopy Fluorescence Issue 104 Single Molecule Microscopy Glass Fluorescence Recovery after Photo-Bleaching 030217 neurology & neurosurgery |
| Zdroj: | Journal of Visualized Experiments : JoVE |
| ISSN: | 1940-087X |
| Popis: | In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues (1). This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light (2). Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode (3,4). They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment. |
| Databáze: | OpenAIRE |
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