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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van de Linde, S., Kasper, R., Heilemann, M. et al. Photoswitching microscopy with standard fluorophores. Appl. Phys. B 93, 725–731 (2008). https://doi.org/10.1007/s00340-008-3250-9
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DOI: https://doi.org/10.1007/s00340-008-3250-9