Photoswitching Mechanism of Cyanine Dyes

Photoswitchable fluorescent probes have been used in recent years to enable super-resolution fluorescence microscopy by single-molecule imaging.1-6 Among these probes are red carbocyanine dyes, which can be reversibly photoconverted between a fluorescent state and a dark state for hundreds of cycles, yielding several thousand detected photons per switching cycle, before permanent photobleaching occurs.7,8 While these properties make them excel-lent probes for super-resolution imaging, the mechanism by which cyanine dyes are photoconverted has yet to be determined. Such an understanding could prove useful for creating new photoswit-chable probes with improved properties. The photoconversion of red cyanine dyes into their dark states occurs upon illumination by red light and is facilitated by a primary thiol in solution,7,9 whereas agents with a secondary thiol do not support photoswitching. These observations suggest that the reactiv