Discrimination of photoblinking and photobleaching on the single molecule level

Wide field observation of individual dye molecules have been performed to study fluorescence intermittency. We demonstrate a data analysis scheme, which enables us to quantify the decay of the ensemble intensity which is due to on/off-blinking of the molecules (photophysical bleaching) by getting rid of the effects of photochemical degradation of the dye (photochemical bleaching). Under the conditions of our experiments, photophysical and thus reversible bleaching is the dominant of the two bleaching mechanisms.