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.

Donor-acceptor Stenhouse adducts (DASAs) are a novel class of solvatochromic photoswitches with increasing importance in photochemistry. Known for their reversibility between open triene and closed cyclized states, these push-pull molecules are applicable in a suite of light-controlled applications. Recent works have sought to understand the DASA photoswitching mechanism and reactive state, as DASAs are vulnerable to irreversible “dark switching” in polar protic solvents. Despite the utility of fluorescence spectroscopy for providing information regarding the electronic structure of organic compounds and gaining mechanistic insight, there have been few studies of DASA fluorescence. Herein, we characterize various photophysical properties of two common DASAs based on Meldrum’s acid and dimethylbarbituric acid by fluorescence spectroscopy. This approach is applied in tandem with complexation by cyclodextrins and cucurbiturils to reveal the zwitterionic charge separation of these photoswitches in aqueous solution and the protective nature of supramolecular complexation against degradative dark switching. DASA-M, for example, was found to form a weak host-guest inclusion complex with (2-hydroxypropyl)-γ-cyclodextrin, with a binding constant K = 60 M−1, but a very strong inclusion complex with cucurbit[7]uril, with K = 27,000 M−1. This complexation within the host cavity was found to increase the half-life of both DASAs in aqueous solution, indicating the significant and potentially useful stabilization of these DASAs by host encapsulation.