Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, USA.
Appl Spectrosc. 2010 Mar;64(3):324-7. doi: 10.1366/000370210790918391.
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.
藻胆蛋白荧光标记物在生物分析中发挥着越来越重要的作用。它们也越来越多地被用作能源研究中的光收集材料。因此，研究这些荧光染料的工作条件至关重要。别藻蓝蛋白（APC）属于藻胆蛋白的一种，具有红色激发和发射特性，因此既是一种有用的荧光团，也是一种光收集材料。本工作通过单分子检测研究了 APC 的饱和辐照度和光漂白。通过提取单分子荧光峰来计算不同激光功率下的平均荧光强度。通过内插平均荧光强度与激发功率的关系图，可以提取实验饱和辐照度。通过比较实验和计算的饱和辐照度，可以证明 APC 的三重态在更高的激发辐照度下形成。应用分子重交叉事件技术研究了 APC 的光漂白。归一化重交叉事件证实，在高激发功率下发生了光漂白。这项工作为 APC 作为荧光团和光收集分子提供了优化的实验条件。
