Photoswitching microscopy with standard fluorophores |
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Authors: | S van de Linde R Kasper M Heilemann M Sauer |
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Institution: | 1.Applied Laser Physics and Laser Spectroscopy and Bielefeld Institute for Biophysics and Nanoscience,Bielefeld University,Bielefeld,Germany |
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Abstract: | We introduce far-field subdiffraction-resolution fluorescence imaging based on photoswitching of individual standard fluorophores in air-saturated solution. Here, photoswitching microscopy relies on the light-induced switching of organic fluorophores (ATTO 655 and ATTO 680) into long-lived metastable dark states and spontaneous repopulation of the fluorescent state. In the presence of low concentrations (2–10 mM) of reducing, thiol-containing compounds such as ß-mercaptoethylamine or glutathione, the density of fluorescent molecules can be adjusted to enable multiple localizations of individual fluorophores with an experimental accuracy of ~20 nm. The method requires wide-field illumination with only a single laser beam for readout and photoswitching and provides superresolution fluorescence images of intracellular structures under live cell compatible conditions. |
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Keywords: | PACS" target="_blank">PACS 32 50 +d 61 05 -a 87 64 M- 87 80 Nj |
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