Department of Biochemistry and Molecular Biophysics, School of Medicine, Washington University in St. Louis, St. Louis, Missouri; Division of Biological Sciences, University of California San Diego, La Jolla, California.
Division of Biological Sciences, University of California San Diego, La Jolla, California.
Biophys J. 2020 Jul 21;119(2):265-273. doi: 10.1016/j.bpj.2020.05.032. Epub 2020 Jun 10.
Analysis of fluctuations arising as fluorescent particles pass through a focused laser beam has enabled quantitative characterization of a broad range of molecular kinetic processes. Two key mathematical frameworks that have enabled these quantifications are fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis. Although these frameworks are effective and accurate when the focused laser beam is well approximated by an infinite Gaussian beam with a waist that is small compared to the size of the region over which the fluorescent particles can diffuse, they cannot be applied to situations in which this region is bounded at the nanoscale. We therefore derived general forms of the FCS and PCH frameworks for bounded systems. The finite-domain form of FCS differs from the classical form in its boundary and initial conditions and requires development of a new Fourier space solution for fitting data. Our finite-domain FCS predicts simulated data accurately and reduces to a previous model for the special case when the system is much larger than the Gaussian beam and can be considered to be infinite. We also derived the PCH form for the bounded systems. Our approach enables estimation of the concentration of diffusing fluorophores within a finite domain for the first time, to our knowledge. The method opens the possibility of quantification of kinetics in several systems for which this has never been possible.
分析荧光粒子穿过聚焦激光束时产生的波动，使对广泛的分子动力学过程进行定量描述成为可能。实现这些量化的两个关键数学框架是荧光相关光谱（FCS）和光子计数直方图（PCH）分析。虽然当聚焦激光束很好地近似为具有与荧光粒子可以扩散的区域的大小相比很小的腰部的无限高斯光束时，这些框架是有效和准确的，但它们不能应用于在纳米尺度上限制该区域的情况。因此，我们为有界系统推导出了 FCS 和 PCH 框架的一般形式。有限域形式的 FCS 在边界和初始条件上与经典形式不同，需要开发用于拟合数据的新傅里叶空间解。我们的有限域 FCS 准确地预测了模拟数据，并且在系统比高斯光束大得多并且可以被认为是无限的特殊情况下简化为先前的模型。我们还推导出了有界系统的 PCH 形式。据我们所知，我们的方法首次能够在有限域内估计扩散荧光团的浓度。该方法为以前不可能进行定量分析的几个系统的动力学提供了可能性。
