Enhancing fluorescence brightness: Effect of reverse intersystem crossing studied by fluorescence fluctuation spectroscopy

Experiments based on fluorescence detection ore limited by the population of the fluorescence marker's long-lived dark triplet state, leading to pronounced photobleaching reactions and blinking which reduces the overage fluorescence signal obtained per time interval. By irradiation with a second red-shifted laser line, we initiate reverse intersystem crossing (ReISC) which enhances the fluorescence signal of common fluorophores up to a factor of 14. The reverse intersystem crossing from the triplet state back to the singlet system is achieved by photoexcitation to higher-excited triplet states, which are, however, prone to photobleaching. We gain insights into the competing pathways of ReISC and photobleaching. The relative efficiencies of these two pathways and the triplet lifetime determine the achievable fluorescence enhancement, which varies strongly with the choice of dye, excitation irradiance and wavelength, and with environmental conditions. The study of ReISC not only results in a better understanding of a fluorescent label's photophysics, but the method is a possible approach to optimize fluorescence emission in experiments, where signal strength is a critical parameter.