Photoswitching microscopy with standard fluorophores

van de Linde, S. and Kasper, R. and Heilemann, M. and Sauer, M. (2008) Photoswitching microscopy with standard fluorophores. Applied Physics B: Lasers and Optics, 93 (4). pp. 725-731. ISSN 0946-2171 (

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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.