Fӧrster Resonance Energy Transfer (FRET) between phycobiliproteins and tandem conjugates
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Single molecule recrossing events was applied to study light tolerance and photobleaching of allophycocyanin (APC), which is one group of phycobiliprotein. Normalized recrossing ratio was defined as Nr/Nt, where Nr was the number of molecules that reenter the probe volume, and Nt was the total number of detected molecules. The ratio of Nr/Nt was regarded as a parameter for light tolerance of the molecules of interest. Normalized molecular recrossings as a function of excitation irradiation showed that the triplet-state formation and photobleaching occurred at high excitation power. Moreover, the similar method was employed to study energy transfer between allophycocyanins and a second conjugated organic dye. The saturation irradiance and triplet states for Streptavidin Allophycocyanin - AlexaFluor-680 (APC-680) was investigated using single molecule recrossings method. By comparing the saturation irradiance and normalized molecular recrossings Nr/Nt for APC and APC-680, it showed that the light tolerance of APC-680 was improved over APC. This improvement resulted from conjugating a second dye to APC, which provided another pathway for energy transfer. The alternative pathway decreased the possibility of photobleaching and triplet state conversion. Therefore, by linking a second dye to phycobiliproteins, the photostability and light tolerance of this system was improved. In order to ease the process of extracting single molecule fluorescence peaks from background noise, the power transform was applied to both simulated and experimental single molecule fluorescence time trace. Power transformations increased the contrast between fluorescence signal bursts and noise. The power transform simplified discerning single molecule signals from background. The results also showed that power transforms had the capability to distinguish the signals that could not be identified in original dataset. The power transform is a simple and reversible method to increase signal to noise ratio of single molecule time traces.