Phycobiliprotein fluorescent labels are playing an increasingly important role in bioanalysis. They are also being used more and more frequently as light-harvesting materials for energy research. It is therefore critical to study the working conditions of these fluorescent dyes. Allophycocyanin (APC) belongs to a group of phycobiliproteins and features red excitation and emission, making it both a useful fluorophore and light-harvesting material. Saturation irradiance and photobleaching of APC were studied by single-molecule detection in this work. The mean fluorescence intensity at different laser powers was calculated from extracted single-molecule fluorescence peaks. By interpolating the figure of the mean fluorescence intensity as a function of excitation power, the experimental saturation irradiance can be extracted. By comparing the experimental with the calculated saturation irradiance, it can be demonstrated that the triplet state for APC was formed at higher excitation irradiance. The technique of molecular recrossing events was applied to investigate the photobleaching of APC. Normalized recrossing events confirmed that photobleaching occurred at high excitation power. This work provided the optimizing experimental conditions for APC both as a fluorophore and as a light-harvesting molecule.
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