Changepoint analysis for single-molecule polarized total internal reflection fluorescence microscopy experiments.
Changepoint analysis for single-molecule polarized total internal reflection fluorescence microscopy experiments.
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DOI：
10.1016/b978-0-12-381270-4.00015-9
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发表时间：
2011
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单个大分子的实验研究为确定其机械化学操作的细节打开了大门。肌球蛋白家族等运动酶一直是此类研究特别有吸引力的目标，部分原因是其中一些酶具有高度加工性，并且它们的“产品”是空间运动。但单分子分辨率有其自身的成本和局限性。通常，观察结果依赖于单个荧光染料分子，这些分子在光漂白之前发射有限数量的光子，并且受到复杂的内部动力学的影响。因此，开发从有限的一组检测到的光子中提取最大有用信息的方法非常重要。我们扩展了一种实验技术，即全内反射荧光显微镜 (polTIRF) 中的多重偏振照明，以记录每个检测到的光子的到达时间和偏振状态。我们还扩展了之前应用于 FRET 实验的分析技术，该技术可以最佳地确定光子发射速率的变化时间。结合这些改进，我们能够以前所未有的细节和时间分辨率来识别分子马达（肌球蛋白 V）的结构动力学。
The experimental study of individual macromolecules has opened a door to determining the details of their mechanochemical operation. Motor enzymes such as the myosin family have been particularly attractive targets for such study, in part because some of them are highly processive and their “product” is spatial motion. But single-molecule resolution comes with its own costs and limitations. Often, the observations rest on single fluorescent dye molecules, which emit a limited number of photons before photobleaching and are subject to complex internal dynamics. Thus, it is important to develop methods that extract the maximum useful information from a finite set of detected photons. We have extended an experimental technique, multiple polarization illumination in total internal reflection fluorescence microscopy (polTIRF), to record the arrival time and polarization state of each individual detected photon. We also extended an analysis technique, previously applied to FRET experiments, that optimally determines times of changes in photon emission rates. Combining these improvements allows us to identify the structural dynamics of a molecular motor (myosin V) with unprecedented detail and temporal resolution.