Department of Biotechnology and Biophysics, Biocenter, Am Hubland, Julius-Maximilians University, 97074 Würzburg, Germany.
Phys Chem Chem Phys. 2013 Jul 7;15(25):10435-45. doi: 10.1039/c3cp50644d. Epub 2013 May 20.
Signal fluctuations in a fluorescence time trace on nanosecond time scales can be induced by specific quenching interactions that report on the dynamics of biomolecules. Fluorescence correlation spectroscopy is an analysis tool to investigate dynamic processes on time scales from pico- to milliseconds or longer. Under certain conditions, e.g. in a solvent of high viscosity, a fluorescence labeled dynamic biomolecule yields multiple independent correlation decays due to rotational and translational diffusion, fluorescence quenching interactions, and fluorophore photophysics. We compared parameter estimation for FCS data with multiple correlation decays by dynamical fingerprint analysis and by the non-linear Levenberg-Marquardt fitting procedure and identified conditions for which dynamical fingerprint analysis can be of advantage. In this context we identified a previously unrecognized photophysical process in ATTO655 that introduces fluorescence intermittency on nanosecond time scales that is absent in MR121. The optimized fitting procedure is used to resolve the viscosity dependence of fluorescence quenching for photoinduced electron transfer probes.
在纳秒时间尺度上，荧光时间轨迹中的信号波动可以由特定的猝灭相互作用引起，这些相互作用可以报告生物分子的动力学。荧光相关光谱学是一种分析工具，可用于研究皮秒到毫秒或更长时间尺度上的动态过程。在某些条件下，例如在高粘度的溶剂中，由于旋转和扩散、荧光猝灭相互作用以及荧光团光物理，荧光标记的动态生物分子会产生多个独立的相关衰减。我们通过动力学指纹分析和非线性 Levenberg-Marquardt 拟合程序比较了具有多个相关衰减的 FCS 数据的参数估计，并确定了动力学指纹分析具有优势的条件。在这方面，我们在 ATTO655 中鉴定了一个以前未被认识到的光物理过程，该过程在纳秒时间尺度上引入了荧光间歇性，而在 MR121 中则不存在。优化的拟合程序用于解决光诱导电子转移探针的荧光猝灭对粘度的依赖性。
