Chen Danni, Li Heng, Yu Bin, Qu Junle
Center for Biomedical Optics and Photonics (CBOP) & College of Physics and Optoelectronic Engineering, Key Laboratory of Optoelectronic Devices and Systems of Guangdong Province and Ministry of Education, Shenzhen University, Shenzhen, 518060, China.
Tsinghua-Berkeley Shenzhen Institute (TBSI), Tsinghua University, Shenzhen, 518055, China.
Nanophotonics. 2022 Mar 16;11(8):1537-1547. doi: 10.1515/nanoph-2021-0681. eCollection 2022 Mar.
Research on dynamic events in living cells, such as intracellular transportation, is important for understanding cell functions. As movements occur within cells, the microenvironment of the moving vesicles or biomacromolecules may affect the behavior of them. Herein, we propose a method of simultaneously monitoring changes in spatial positions and the local environment related to the fluorescence lifetime, i.e., four-dimensional (4D) multi-particle parallel-tracking in living cells. Based on double-helix point spread function (DH-PSF) microscopy and streak camera, the method combines three-dimensional (3D) localization methods and fluorescence lifetime imaging. By modifying the PSF of the system, the 3D positions and fluorescence lifetime information for several molecules within a depth of a few microns can be acquired simultaneously from a single snapshot. The feasibility of this method is verified by simulating the real-time tracking of a single particle with a given trajectory. In addition, a proof-of-concept 4D tracking system based on the DH-PSF and streak camera was built. The experimental results show that the 3D localization and lifetime precision are (, , ) = (26 nm, 35 nm, 53 nm) and () = 103 ps, respectively, and the effective depth of field is approximately 4 μm. Finally, intracellular endocytosis in a living cell was observed using the system, which demonstrated the successful 4D tracking of two microspheres moving within an axial depth of 4 μm. This work opens a new perspective for research of dynamic processes, by providing information about the chemical (microenvironments) and physical (positions) changes of moving targets in living cells.
对活细胞中的动态事件进行研究，如细胞内运输，对于理解细胞功能至关重要。由于细胞内会发生各种运动，移动的囊泡或生物大分子的微环境可能会影响它们的行为。在此，我们提出了一种同时监测与荧光寿命相关的空间位置和局部环境变化的方法，即活细胞中的四维（4D）多粒子平行跟踪。基于双螺旋点扩散函数（DH-PSF）显微镜和条纹相机，该方法将三维（3D）定位方法与荧光寿命成像相结合。通过修改系统的点扩散函数，可以从单个快照中同时获取几微米深度范围内多个分子的3D位置和荧光寿命信息。通过模拟具有给定轨迹的单个粒子的实时跟踪，验证了该方法的可行性。此外，构建了基于DH-PSF和条纹相机的概念验证4D跟踪系统。实验结果表明，3D定位和寿命精度分别为（x，y，z）=（26 nm，35 nm，53 nm）和（τ）= 103 ps，有效景深约为4μm。最后，使用该系统观察了活细胞中的细胞内吞作用，成功实现了对两个在4μm轴向深度内移动的微球的4D跟踪。这项工作通过提供有关活细胞中移动目标的化学（微环境）和物理（位置）变化的信息，为动态过程的研究开辟了新的视角。
