Non-exponential Kinetics of Photoblinking and Photobleaching of Rhodamine 6G in Polyvinylalcohol

Photobleaching and photoblinking have proven to be the main bottleneck for single-molecule microscopy and spectroscopy at room temperature. Here, a quantitative ensemble study of the kinetics of photoblinking and photobleaching at room temperature of a typical fluorescent label, Rhodamine 6G, in a polar, hydrogen bonding, solid matrix of polyvinylalcohol is presented as a function of the excitation intensity and the presence of oxygen. To achieve uniform irradiation of all molecules present in the excitation focus, the sample (2.0 x 10^–5 M R6G in PVA spin-coated on a quartz substrate) is covered by a pinhole array mask with holes of diameter 40 ∝m, each addressable as an individual sample. The experiments are performed at intensities between 65 mW/cm² and 320 W/cm² and the measured emissivity of the system is normalized to that at 65 mW/cm². The emissivity is shown to decrease by a factor of up to 20 at high intensity indicating the presence of a dark state, which would lead to photoblinking of single molecules. The triplet state of R6G cannot be this dark state, as it is hardly populated at excitation intensities below 1 kW/cm². However, our data suggest that this state might be an intermediate between the singlet excited state and the dark state, which could be for instance a radical. Fig. 1 shows long-term fluorescence traces obtained at room temperature in an air atmosphere, displaying photobleaching. The rates governing blinking and bleaching are found to be widely distributed. Bleaching is shown to be more efficient in air, while blinking is more pronounced in the nitrogen atmosphere, because the lifetime of the dark state becomes longer.