Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, Wollongong, Australia.
Spectroscopy of Soft Matter, University of Bayreuth, Bayreuth, Germany.
Biophys J. 2019 Sep 3;117(5):950-961. doi: 10.1016/j.bpj.2019.07.015. Epub 2019 Jul 19.
Understanding how multiprotein complexes function in cells requires detailed quantitative understanding of their association and dissociation kinetics. Analysis of the heterogeneity of binding lifetimes enables the interrogation of the various intermediate states formed during the reaction. Single-molecule fluorescence imaging permits the measurement of reaction kinetics inside living organisms with minimal perturbation. However, poor photophysical properties of fluorescent probes limit the dynamic range and accuracy of measurements of off rates in live cells. Time-lapse single-molecule fluorescence imaging can partially overcome the limits of photobleaching; however, limitations of this technique remain uncharacterized. Here, we present a structured analysis of which timescales are most accessible using the time-lapse imaging approach and explore uncertainties in determining kinetic subpopulations. We demonstrate the effect of shot noise on the precision of the measurements as well as the resolution and dynamic range limits that are inherent to the method. Our work provides a convenient implementation to determine theoretical errors from measurements and to support interpretation of experimental data.
理解多蛋白复合物在细胞中的功能需要对其结合和解离动力学有详细的定量了解。分析结合寿命的异质性可以探究反应过程中形成的各种中间状态。单分子荧光成像允许在最小干扰的情况下在活生物体内部测量反应动力学。然而，荧光探针的光物理性质较差限制了活细胞中离解速率测量的动态范围和准确性。延时单分子荧光成像可以部分克服光漂白的限制；然而，该技术的局限性尚未得到明确。在这里，我们对延时成像方法可用于测量的时间尺度进行了结构化分析，并探讨了确定动力学亚群时的不确定性。我们展示了拍摄噪声对测量精度的影响，以及该方法固有的分辨率和动态范围限制。我们的工作提供了一种方便的实现方法，可从测量中确定理论误差，并支持对实验数据的解释。
