利用三维单分子荧光跟踪技术研究单细胞内三维扩散分布及凋亡的影响

生物大分子的扩散支配了细胞内的生命活动，也反映了细胞内拥挤微环境的特点，因此细胞内扩散速度的准确测量在长久以来都受到研究者的关注。但是，由于传统方法仅能每次测量细胞上单个位点的扩散，目前人们并不清楚生物大分子如何在真实的细胞内三维空间中进行扩散,特别是扩散在细胞内的三维分布。在本项目中，我们将在过去二维扩散测量技术的基础上创立一种快速测量单细胞内三维扩散分布的新方法，即通过搭建轴向测量和物镜锁焦装置，建立三维单分子荧光跟踪平台，然后由细胞内的单分子荧光运动轨迹，直接计算单细胞内的三维扩散分布。利用该技术，我们将研究凋亡对细胞内三维扩散分布的影响，以及线粒体释放凋亡信号分子和凋亡小体形成过程的局部扩散特征。我们将通过对三维扩散分布的精确测量来阐明凋亡细胞内生物大分子的动力学特点，并深入分析凋亡对细胞内微环境的影响，从而在生物物理角度进一步理解细胞的凋亡机制。

Not only the intracellular macromolecule diffusing dominates the cellular activities, but also reflects the characteristic of the crowding micro-environments in cells. Therefore, the how to accurately measure the intracellular diffusion coefficients is always drawing attention to the scientific researchers. However, due to the limitation of the traditional techniques by which only the diffusion coefficient of one point in the cell could be measured once, until now there is no information about the diffusion of macromolecule in the truly three-dimensional (3D) space in cells, and particularly the 3D diffusion map. In the research proposal, we aim to develop a novel method to map rapidly the 3D diffusion distribution in single cells, based on our previously two-dimensional mapping method. To this end, we will build 3D single-molecule fluorescence tracking platform, by setting up the z-axis detecting and objective focus locking equipments. And from the 3D trajectories of the single fluorescence molecules in the cell, the 3D diffusion map of the single cell can be calculated directly. Using this method, we will study the 3D diffusion map in apoptotic cells, and focus on the local diffusion characteristic around the mitochondria when releasing the signal molecules, and that during the formation of apoptotic bodies. By using the precise measurement of the 3D diffusion map, we will elucidate the dynamic characteristic of the intracellular macromolecules during apoptosis, and analyze the effect of apoptosis on the intracellular micro-environments. This work will shed new light on the mechanism of apoptosis in the view of biophysics.