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

Abstract

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.

Harnessing Hypoxia‐Dependent Cyanine Photocages for In Vivo Precision Drug Release

“…The reference Cy‐C without a secondary amine group further demonstrated that the amino group as electron donor plays the key role for the PeT process. This proposed radical‐mediated photolysis is consistent with the reported cyanines [10f] . Finally, with the help of water as a reactant, the photolysis fragments H‐Cy are obtained.…”

Turn-on mode diarylethenes for bioconjugation and fluorescence microscopy of cellular structures

“…dyes routinely used for superresolution STORM. By contrast, our fDAEs do not require strict chemical control of the imaging conditions (48,49). The Urbach tail absorption of the OFs allowed us to apply just a 532-or 561-nm laser in order to induce single-molecule photoswitching (blinking) and acquire superresolution images.…”

Targetable Conformationally Restricted Cyanines Enable Photon‐Count‐Limited Applications**

“…We previously reported that these conformationally restricted cyanines do not undergo efficient blinking under conventional high‐thiol d STORM conditions. Instead, these probes exhibit improved recovery from NaBH 4 reduction, providing high‐quality SMLM imaging when appended to a small molecule targeting agent [3a, 27] …”