Light harvesting and energy transfer between allophycocyanin (APC) and a tandem conjugate dye was investigated using single-molecule recrossings in the probe volume. By comparing saturation irradiance and recrossing events of APC and the tandem conjugate Streptavidin Allophycocyanin–AlexaFluor-680 (APC-680), it was demonstrated that the light tolerance and photostability of APC-680 were improved over APC. This improvement was due to conjugating a tandem dye to APC, which provided an alternative pathway for energy transfer. This additional pathway decreases photobleaching and triplet-state conversion. In this study, the average fluorescence intensity at different laser powers was calculated from extracted signal peaks. Saturation irradiance was determined by interpolating the average intensity as a function of excitation power. By comparing the saturation irradiance of APC and APC-680, it was demonstrated that light tolerance was not significantly increased by having a small fluorophore covalently linked to the protein. The molecular recrossing method was used to determine triplet-state conversion and photobleaching effects. The number of molecules that were detected within 2 ms of each other was defined as the number of recrossing events (<i>N</i><sub>r</sub>). The total number of molecules detected was defined as <i>N</i><sub>t</sub>. The normalized number of recrossings events (<i>N</i><sub>r</sub>/<i>N</i><sub>t</sub>) showed that energy transfer between APC and AlexaFluor-680 could reduce triplet-state formation of phycobiliproteins. In this case, the saturation irradiance was not improved, but the resistance to photobleaching and triplet-state conversion was improved.
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