Mechanisms of photobleaching investigated by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) can be used to investigate the photobleaching properties of fluorophores in solution. The advantage with this method is that in addition to the photobleaching rate the formation and decay rates of the triplet state can be measured. In this way, it is possible to calculate the photodestruction quantum yield and relate the photostability of a fluorescent compound in a certain environment to the photodynamical behaviour of the singlet-triplet transitions. This is likely to contribute to a better understanding of the mechanisms of photobleaching given the central importance of dye triplet states in photobleaching processes. The approach was applied to the measurement and characterization of the photobleaching of Rh6G in aqueous solution and FITC in 1 mM sodium carbonate buffer (pH 9). The photobleaching yields measured are discussed in view of the simultaneous triplet properties at different excitation intensities, oxygen concentrations as well as in the presence or absence of quencher molecules. This study suggests that FCS is likely to provide a valuable tool for the elucidation of the mechanisms of photobleaching, which are far from understood in all their details.