Identification of a thermally activated process in the Cy3 photobleaching mechanism.

Single molecule experiments were used to investigate the photobleaching mechanism of single Cy3 molecules encapsulated in large unilamellar vesicles. Analysis of thousands of individual fluorescence traces evidenced that the survival probability distribution is monoexponential with an average survival time depending on the laser intensity, the concentration of molecular oxygen, the temperature and the rigidity of the medium. Taken together, our data suggest that the photobleaching of Cy3 molecules is governed by two parallel pathways, namely photooxidation and a thermally activated structural rearrangement of the Cy3 molecules in their excited state.