Mechanisms of high-order photobleaching and its relationship to intracellular ablation.
Mechanisms of high-order photobleaching and its relationship to intracellular ablation.
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DOI：
10.1364/boe.2.000816
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发表时间：
2011-03-04
影响因子：
3.4
通讯作者：
Heisterkamp A
中科院分区：
文献类型：
--
作者：
Kalies S;Kuetemeyer K;Heisterkamp A
在双光子激光扫描显微术中，飞秒激光脉冲的光漂白速率对激发功率的依赖关系可能具有二次、三次甚至双二次的顺序。迄今为止，关于这种所谓的高阶光漂白仍有许多悬而未决的问题。我们研究了在双光子显微镜中细胞环境中的内在（增强型绿色荧光蛋白（eGFP））和外在（Hoechst 33342）荧光团的光漂白动力学。此外，我们研究了漂白和活性氧物种的形成之间的相关性。我们观察到eGFP的漂白阶数为3和4，Hoechst的漂白阶数为2和3，在一定波长下逐步增加。随着时间的推移与漂白相关的活性氧物种的增加被确认。将我们的结果与细胞内消融中涉及的相互作用光子的量和所涉及的能量状态的机制进行比较，我们发现在这两种情况下都形成了低密度等离子体，并且在两者之间具有平滑的过渡。光漂白，然而，介导的顺序吸收和多光子电离，而消融是由后者和级联电离过程占主导地位。
In two-photon laser-scanning microscopy using femtosecond laser pulses, the dependence of the photobleaching rate on excitation power may have a quadratic, cubic or even biquadratic order. To date, there are still many open questions concerning this so-called high-order photobleaching. We studied the photobleaching kinetics of an intrinsic (enhanced Green Fluorescent Protein (eGFP)) and an extrinsic (Hoechst 33342) fluorophore in a cellular environment in two-photon microscopy. Furthermore, we examined the correlation between bleaching and the formation of reactive oxygen species. We observed bleaching-orders of three and four for eGFP and two and three for Hoechst increasing step-wise at a certain wavelength. An increase of reactive oxygen species correlating with the bleaching over time was recognized. Comparing our results to the mechanisms involved in intracellular ablation with respect to the amount of interacting photons and involved energetic states, we found that a low-density plasma is formed in both cases with a smooth transition in between. Photobleaching, however, is mediated by sequential-absorption and multiphoton-ionization, while ablation is dominated by the latter and cascade-ionization processes.