Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation

Fluorescence spectroscopic measurements at the single-molecule level usually require large absorption cross sections and fluorescence quantum yields for the dyes under study. In addition to these parameters, the collectable number of fluorescence photons and, thus, the signal-to-noise ratio of the measurement, is influenced by processes like triplet-state population and photobleaching, shifting the saturation threshold of the dye to lower excitation intensities. Confocal fluorescence correlation spectroscopy (FCS) is a versatile method to precisely determine photon emission rates of single molecules but also gives access to rate constants of dynamic bleaching and intersystem crossing. In recent FCS studies in solution and living cells, two-photon excitation with its inherent spatial sectioning has proven to be a very valuable alternative to minimize background and cumulative signal loss. However, there is evidence that in many dye systems, the photobleaching rates with two-photon excitation are significantly enhanced with respect to one-photon excitation at comparable photon-emission yields. The reasons have so far remained mainly speculative. In the present study, potential photobleaching pathways are investigated by adding chemical stabilizers and by working at different oxygen concentrations. The results suggest that the population of triplet states does not appear to be responsible for the limited emission rate with two-photon excitation. Rather, photobleaching pathways via the formation of radicals seem to be plausible causes for the signal limitation. Favorable conditions are discussed to maximize the overall photon-collection yield in two-photon experiments.