Jazani Sina, Xu 徐伟青 Lance W Q, Sgouralis Ioannis, Shepherd Douglas P, Pressé Steve
Department of Biophysics and Biophysical Chemistry, Johns Hopkins Medicine, Baltimore.
Center for Biological Physics, Department of Physics, Arizona State University, Tempe.
ACS Photonics. 2022 Jul 20;9(7):2489-2498. doi: 10.1021/acsphotonics.2c00614. Epub 2022 Jul 7.
Tracking single molecules continues to provide new insights into the fundamental rules governing biological function. Despite continued technical advances in fluorescent and non-fluorescent labeling as well as data analysis, direct observations of trajectories and interactions of multiple molecules in dense environments remain aspirational goals. While confocal methods provide a means to deduce dynamical parameters with high temporal resolution, such as diffusion coefficients, they do so at the expense of spatial resolution. Indeed, on account of a confocal volume's symmetry, typically only distances from the center of the confocal spot can be deduced. Motivated by the need for true three dimensional high speed tracking in densely labeled environments, we propose a computational tool for tracking many fluorescent molecules traversing multiple, closely spaced, confocal measurement volumes providing independent observations. Various realizations of this multiple confocal volumes strategy have previously been used for long term, large area, tracking of one fluorescent molecule in three dimensions. What is more, we achieve tracking by directly using single photon arrival times to inform our likelihood and exploit Hamiltonian Monte Carlo to efficiently sample trajectories from our posterior within a Bayesian nonparametric paradigm. A nonparametric paradigm here is warranted as the number of molecules present are, themselves, unknown. Taken together, we provide a computational framework to infer trajectories of multiple molecules at once, below the diffraction limit (the width of a confocal spot), in three dimensions at sub-millisecond or faster time scales.
追踪单个分子不断为理解生物功能的基本规则提供新的见解。尽管在荧光和非荧光标记以及数据分析方面技术不断进步，但在密集环境中对多个分子的轨迹和相互作用进行直接观察仍然是理想目标。虽然共聚焦方法提供了一种以高时间分辨率推导动力学参数（如扩散系数）的手段，但这样做是以牺牲空间分辨率为代价的。实际上，由于共聚焦体积的对称性，通常只能推导出与共聚焦光斑中心的距离。出于在密集标记环境中进行真正三维高速追踪的需求，我们提出了一种计算工具，用于追踪许多穿过多个紧密间隔的共聚焦测量体积的荧光分子，这些体积提供独立的观测。这种多共聚焦体积策略的各种实现方式此前已用于在三维空间中对单个荧光分子进行长期、大面积的追踪。此外，我们通过直接利用单光子到达时间来确定似然性，并利用哈密顿蒙特卡罗方法在贝叶斯非参数范式内从后验中高效地采样轨迹来实现追踪。这里采用非参数范式是因为存在的分子数量本身是未知的。综上所述，我们提供了一个计算框架，能够在亚毫秒或更快的时间尺度上，在三维空间中同时推断低于衍射极限（共聚焦光斑的宽度）的多个分子的轨迹。
