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摘要: 单颗粒示踪（Single particle tracking，SPT）技术是应用显微镜系统对细胞内单个特定荧光或散射颗粒的定位和追踪。由于SPT能够实时监控活细胞内复杂、高度动态的组织结构的变化并提供结构—功能间的动力学关系，因此在细胞生物学上有重要的应用。本文总结了SPT的机理以及在细胞上的应用，首先介绍了SPT的动力学原理，包括单颗粒定位，轨道重建以及轨道分析，然后总结了SPT技术现阶段重点发展的光学材料及仪器，最后阐述了SPT在细胞膜、细胞内信号通路、分子转运机制、遗传信息表达以及病毒感染机制的应用。此外，本文还对SPT技术未来的发展进行了展望。Abstract: Single particle tracking(SPT) technique locates and tracks individual fluorescent or scattering particles within a cell with the help of microscope system. Based on the ability of real-time monitoring of the complex and highly dynamic changes in tissue structure within living cells and the ability to provide dynamic relationships between structure and function, SPT has important applications in cell biology. In this review, the mechanism of SPT and its application on cells are summarized. Firstly, the dynamics of SPT are introduced, including single particle localization, trajectory reconstruction and analysis. Then the optical materials and instruments that SPT technology focuses on at the present stage are described. Finally, the application of SPT in cell membrane, intracellular signaling pathway, molecular transport mechanism, genetic information expression, and viral infection mechanism are proposed. In addition, the advance of SPT technology are prospected in this paper.
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Key words:
- SPT technology /
- cell /
- dynamics /
- optical materials and instruments
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图 1 SPT轨迹重建示意图[27]。(a)借助PSF确定颗粒的位置; (b)只和邻近的点进行连接; (c)按照时间顺序连接形成一个轨迹; (d)对轨迹进行数据分析，提取出动力学信息
Figure 1. Schematic representation of SPT[27]. (a)Particles are localized by finding the central locations of their point spread functions; (b)localizations belonging to the same particle at different times are connected using algorithms such as nearest neighbor; (c)chronological series of linked localizations form a time series trajectory; (d)dynamic information is extracted based on a statistical analysis of the trajectories
图 4 细胞膜的分层结构[17]。(a)膜隔室层，由肌动蛋白骨架分隔整个细胞膜形成，并且跨膜蛋白锚定在肌动蛋白骨架上; (b)“筏域”, 富含胆固醇，尺寸受到膜隔室的限制; (c)由膜相关蛋白以及整合蛋白的低聚物组成，存在时间非常短
Figure 4. Three-tiered hierarchical structure of mesoscale domains in the plasma membrane[17]. (a)Membrane compartments which stem from the partitioning of the entire plasma membrane by the membrane associated actin-based membrane skeleton(fence) and TM proteins anchored to the membrane skeleton fence(pickets); (b)cholesterol-containing raft domains, with sizes limited by the membrane compartments; (c)dimers and greater oligomers of membane associated and integral membrane proteins, which might exist only transiently
图 5 单个QD-驱动蛋白在活细胞内的运动[20]。(a)Hela细胞的明场图像；(b)将连续的600帧图像叠加得到的最终图像，线性轨迹表示单个QD-驱动蛋白做定向运动(实心箭头)，空心箭头表示的是其他一些QD-驱动蛋白做随机运动
Figure 5. Single QD-kinesin motions in a living cell[20]. (a)Bright-field image of a HeLa cell; (b)image obtained by superimposing the 600 consecutives frames in the image sequence. The linear traject-ories are indicative of directed motions of individual QD-kinesin. Examples are marked by the full arrows. The trajectories of diffusing QD-Ks(marked by empty arrowheads) have a random shape in the superimposed image
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