Single molecule photobleaching: increasing photon yield and survival time through suppression of two-step photolysis

Irreversible photobleaching imparts the unavoidable limitation on single-molecule spectroscopy: when the fluorescent probe molecule becomes nonfluorescent, the experiment is over. A detailed study of the photobleaching rate for rhodamine 6G under vacuum reveals that low excitation rates yield long photochemical survival times and unprecedented numbers of emitted photons. A four-level system is used to model the photobleaching rate, reproducing the experimental bleaching rate and photon yield from single molecule and ensemble experiments. A higher triplet excited state Tn is found to be the predominant reactive state for photobleaching. This model produces a calculated quantum yield for photobleaching on the order of 7×10−7 bleaching events per excitation to Tn.