Photoinduced formation of reversible dye radicals and their impact on super-resolution imaging

Photochem Photobiol Sci. 2011 Apr;10(4):499-506. doi: 10.1039/c0pp00317d. Epub 2010 Dec 10.


Radical ions of organic dyes are highly reactive species and have been studied for decades by transient absorption spectroscopy and pulse radiolysis experiments in oxygen-depleted solution. Here we show by continuous wave EPR, absorption, and fluorescence experiments that the triplet state of rhodamine dyes can be photoreduced by thiols to form stable radical anions in aqueous solution with a lifetime of up to several hours. Our data demonstrate that reduction of the triplet state and photoinduced oxidation of reactive intermediates by oxygen represents a general mechanism for reversible photoswitching of dyes in aqueous thiol-containing solutions highlighting the key role of molecular oxygen for super-resolution fluorescence imaging. Since cells contain the thiol glutathione at millimolar concentrations and reactive oxygen species are formed as side products our findings are of consequence for live cell fluorescence microscopy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Electron Spin Resonance Spectroscopy
  • Fluorescent Dyes / chemistry*
  • Fluorescent Dyes / radiation effects
  • Free Radicals / chemistry
  • Free Radicals / metabolism*
  • Glutathione / chemistry
  • Light
  • Magnetics
  • Microscopy, Fluorescence
  • Oxidation-Reduction
  • Rhodamines / chemistry*
  • Rhodamines / radiation effects
  • Sulfhydryl Compounds / chemistry


  • Fluorescent Dyes
  • Free Radicals
  • Rhodamines
  • Sulfhydryl Compounds
  • Glutathione