Unifying Mechanism for Thiol-Induced Photoswitching and Photostability of Cyanine Dyes

J Am Chem Soc. 2020 Jul 22;142(29):12681-12689. doi: 10.1021/jacs.0c03786. Epub 2020 Jul 13.


Cyanines (Cy3, Cy5, Cy3B) are the most utilized dyes for single-molecule fluorescence and localization-based super-resolution imaging. These modalities exploit cyanines' versatile photochemical behavior with thiols. A mechanism reconciling seemingly divergent results and enabling control over cyanine photoreactivity is however missing. Utilizing single-molecule fluorescence on Cy5 and Cy5B, transient-absorption spectroscopy, and DFT modeling on a range of cyanine dyes, herein we show that photoinduced electron transfer (PeT) from a thiolate to Cy in their triplet excited state and then triplet-to-singlet intersystem crossing in the nascent geminate radical pair are crucial steps. Next, a bifurcation occurs, yielding either back electron transfer and regeneration of ground state Cy, required for photostabilization, or Cy-thiol adduct formation, necessary for super-resolution microscopy. Cy regeneration via photoinduced thiol elimination is favored by adduct absorption spectra broadening. Elimination is also shown to occur through an acid-catalyzed reaction. Overall, our work provides a roadmap for designing fluorophores, photoswitching agents, and triplet excited state quenchers for single-molecule and super-resolution imaging.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbocyanines / chemistry*
  • Density Functional Theory
  • Fluorescent Dyes / chemistry*
  • Molecular Structure
  • Photochemical Processes
  • Sulfhydryl Compounds / chemistry*


  • Carbocyanines
  • Fluorescent Dyes
  • Sulfhydryl Compounds