Eggeling Christian, Volkmer Andreas, Seidel Claus A M
Max-Planck-Institute for Biophysical Chemistry, Department of Nanobiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.
Chemphyschem. 2005 May;6(5):791-804. doi: 10.1002/cphc.200400509.
Under high-excitation irradiance conditions in one- and two-photon induced fluorescence microscopy, the photostability of fluorescent dyes is of crucial importance for the detection sensitivity of single molecules and for the contrast in fluorescence imaging. Herein, we report on the dependence of photobleaching on the excitation conditions, using the dye Rhodamine 6G as a typical example. The different excitation modes investigated include 1) one-photon excitation into the first-excited singlet state in the range of 500 to 528 nm by continuous wave and picosecond-pulsed lasers and 2) two- and one-photon excitation to higher-excited singlet states at 800 and 350 nm, respectively, by femtosecond pulses. Experimental strategies are presented, which allow resolving the photophysics. From single-molecule trajectories and fluorescence correlation spectroscopy, as well as with a simple theoretical model based on steady-state solutions of molecular rate equation analysis, we determined the underlying photobleaching mechanisms and quantified the photokinetic parameters describing the dependence of the fluorescence signal on the excitation irradiance. The comparison with experimental data and an exact theoretical model show that only minor deviations between the different theoretical approaches can be observed for high-pulsed excitation irradiances. It is shown that fluorescence excitation is in all cases limited by photolysis from higher-excited electronic states. In contrast to picosecond-pulsed excitation, this is extremely severe for both one- and two-photon excitation with femtosecond pulses. Furthermore, the photostability of the higher-excited electronic states is strongly influenced by environmental conditions, such as polarity and temperature.
在单光子和双光子诱导荧光显微镜的高激发辐照条件下,荧光染料的光稳定性对于单分子检测灵敏度和荧光成像对比度至关重要。在此,我们以罗丹明6G染料为例,报告光漂白对激发条件的依赖性。所研究的不同激发模式包括:1)通过连续波和皮秒脉冲激光在500至528nm范围内单光子激发到第一激发单重态;2)通过飞秒脉冲分别在800nm和350nm处双光子和单光子激发到更高激发单重态。本文提出了一些实验策略,可用于解析光物理过程。通过单分子轨迹和荧光相关光谱,以及基于分子速率方程分析稳态解的简单理论模型,我们确定了潜在的光漂白机制,并量化了描述荧光信号对激发辐照依赖性的光动力学参数。与实验数据和精确理论模型的比较表明,对于高脉冲激发辐照,不同理论方法之间仅存在微小偏差。结果表明,在所有情况下,荧光激发均受限于来自更高激发电子态的光解。与皮秒脉冲激发相比,飞秒脉冲的单光子和双光子激发的这种情况极为严重。此外,更高激发电子态的光稳定性受极性和温度等环境条件的强烈影响。