Photoswitching microscopy with standard fluorophores

S. van de Linde, R. Kasper, M. Heilemann, M. Sauer

Research output: Contribution to journalArticlepeer-review

89 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)725-731
Number of pages7
JournalApplied Physics B: Lasers and Optics
Volume93
Issue number4
DOIs
Publication statusPublished - 19 Oct 2008

Keywords

  • photoswitching
  • microscopy
  • flurophores
  • subdiffraction-resolution
  • intracellular structures

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