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3D single-molecule tracking using one- and two-photon excitation microscopy

机译:使用一光子激发和两光子激发显微镜进行3D单分子跟踪

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Three dimensional single-molecule tracking (3D-SMT) has revolutionized the way we study fundamental cellular processes. By analyzing the spatial trajectories of individual molecules (e.g. a receptor or a signaling molecule) in 3D space, one can discern the internalization or transport dynamics of these molecules, study the heterogeneity of subcellular structures, and elucidate the complex spatiotemporal regulation mechanisms. Sub-diffraction localization precision, sub-millisecond temporal resolution and tens-of-seconds observation period are the benchmarks of current 3D-SMT techniques. We have recently built two molecular tracking systems in our labs. The first system is a previously reported confocal tracking system, which we denote as the 1P-1E-4D (one-photon excitation, one excitation beam, and four fiber-coupled detectors) system. The second system is a whole new design that is based on two-photon excitation, which we denote as the 2P-4E-1D (two-photon excitation, four excitation beams, and only one detector) system. Here we compare these two systems based on Monte Carlo simulation of tracking a diffusing fluorescent molecule. Through our simulation, we have characterized the limitation of individual systems and optimized the system parameters such as magnification, z-plane separation, and feedback gains.
机译:三维单分子跟踪(3D-SMT)彻底改变了我们研究基本细胞过程的方式。通过分析3D空间中单个分子(例如受体或信号分子)的空间轨迹,可以识别这些分子的内在化或转运动力学,研究亚细胞结构的异质性,并阐明复杂的时空调节机制。亚衍射定位精度,亚毫秒级时间分辨率和数十秒观察周期是当前3D-SMT技术的基准。我们最近在实验室中建立了两个分子跟踪系统。第一个系统是先前报道的共聚焦跟踪系统,我们将其称为1P-1E-4D(单光子激发,一个激发束和四个光纤耦合探测器)系统。第二个系统是基于双光子激发的全新设计,我们将其称为2P-4E-1D(双光子激发,四个激发光束和仅一个检测器)系统。在这里,我们基于跟踪散射荧光分子的蒙特卡罗模拟比较了这两个系统。通过我们的仿真,我们确定了单个系统的局限性,并优化了系统参数,例如放大倍数,z平面分离和反馈增益。

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