Ringemann Christian, Schönle Andreas, Giske Arnold, von Middendorff Claas, Hell Stefan W, Eggeling Christian
Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.
Chemphyschem. 2008 Mar 14;9(4):612-24. doi: 10.1002/cphc.200700596.
Experiments based on fluorescence detection are limited by the population of the fluorescence marker's long-lived dark triplet state, leading to pronounced photobleaching reactions and blinking which reduces the average fluorescence signal obtained per time interval. By irradiation with a second, red-shifted laser line, we initiate reverse intersystem crossing (ReISC) which enhances the fluorescence signal of common fluorophores up to a factor of 14. The reverse intersystem crossing from the triplet state back to the singlet system is achieved by photoexcitation to higher-excited triplet states, which are, however, prone to photobleaching. We gain insights into the competing pathways of ReISC and photobleaching. The relative efficiencies of these two pathways and the triplet lifetime determine the achievable fluorescence enhancement, which varies strongly with the choice of dye, excitation irradiance and wavelength, and with environmental conditions. The study of ReISC not only results in a better understanding of a fluorescent label's photophysics, but the method is a possible approach to optimize fluorescence emission in experiments, where signal strength is a critical parameter.
基于荧光检测的实验受到荧光标记物长寿命暗三重态数量的限制，导致明显的光漂白反应和闪烁现象，从而降低了每个时间间隔内获得的平均荧光信号。通过用第二条红移激光线进行照射，我们启动了反向系间窜越（ReISC），这可将常见荧光团的荧光信号增强至14倍。从三重态回到单重态系统的反向系间窜越是通过光激发到更高激发的三重态来实现的，然而，这些更高激发的三重态容易发生光漂白。我们深入了解了反向系间窜越和光漂白的竞争途径。这两种途径的相对效率和三重态寿命决定了可实现的荧光增强效果，其会因染料的选择、激发辐照度和波长以及环境条件而有很大差异。对反向系间窜越的研究不仅有助于更好地理解荧光标记物的光物理性质，而且该方法是在信号强度是关键参数的实验中优化荧光发射的一种可能途径。
