National Institute of Standards and Technology and University of Colorado, JILA, Boulder, Colorado 80309, United States.
Department of Chemistry, University of Colorado, Boulder, Colorado 80309, United States.
J Phys Chem B. 2021 Jun 17;125(23):6080-6089. doi: 10.1021/acs.jpcb.1c01036. Epub 2021 Jun 7.
Single-molecule fluorescence resonance energy transfer (smFRET) experiments permit detailed examination of microscopic dynamics. However, kinetic rate constants determined by smFRET are susceptible to systematic underestimation when the rate constants are comparable to the data acquisition rate. We demonstrate how such systematic errors in camera-based total internal reflection fluorescence microscopy experiments can be greatly reduced by using stroboscopic illumination/detection, allowing accurate rate constant determination up to the data sampling rate and yielding an order of magnitude increase in the dynamic range. Implementation of these stroboscopic smFRET ideas is straightforward, and the stroboscopically obtained data are compatible with multiple trajectory analysis methods, including dwell-time analysis and hidden Markov modeling. Such stroboscopic methods therefore offer a remarkably simple yet valuable addition to the smFRET toolkit, requiring only relatively modest modification to the normal data collection and analysis procedures.
单分子荧光共振能量转移 (smFRET) 实验可详细检测微观动力学。然而，当速率常数与数据采集速率相当时，smFRET 确定的动力学速率常数容易受到系统低估。我们展示了如何通过使用频闪照明/检测来大大降低基于相机的全内反射荧光显微镜实验中的这种系统误差，从而可以在高达数据采样率的范围内准确确定速率常数，并使动态范围提高一个数量级。这些频闪 smFRET 思想的实现非常简单，并且频闪获得的数据与多种轨迹分析方法兼容，包括停留时间分析和隐马尔可夫建模。因此，这种频闪方法为 smFRET 工具包提供了一种非常简单但非常有价值的补充，仅需要对正常的数据采集和分析过程进行相对较小的修改。
