Bezzi Paola, Volterra Andrea
Cold Spring Harb Protoc. 2014 May 1;2014(5):pdb.prot081711. doi: 10.1101/pdb.prot081711.
Optical imaging techniques are well suited for following the dynamics of physiological processes in living cells. Total internal reflection fluorescence (TIRF) microscopy based on evanescent wave illumination (EWi) allows spectacular, real-time visualization of individual vesicle movements, fusions, and retrievals at the cell surface (i.e., within 100 nm of the plasma membrane). TIRF microscopy is an ideal approach for studying the properties of exocytosis and recycling in cultured astrocytes, particularly because these cells have a rather flat surface and contain secretory vesicles with sparse distribution. Among all populations of secretory vesicles, we focus here on synaptic-like microvesicles (SLMVs). We illustrate how TIRF microscopy using EWi is useful to study exocytosis and recycling of SLMVs at the single-vesicle level and, when combined with epifluorescence illumination (EPIi), can provide detailed information on the kinetics of exocytosis, endocytosis, and re-acidification at the whole-cell level.
光学成像技术非常适合用于追踪活细胞中生理过程的动态变化。基于倏逝波照明（EWi）的全内反射荧光（TIRF）显微镜能够对细胞表面（即质膜100纳米范围内）单个囊泡的运动、融合和回收进行惊人的实时可视化观察。TIRF显微镜是研究培养星形胶质细胞中胞吐作用和再循环特性的理想方法，特别是因为这些细胞具有相当平坦的表面，并且含有分布稀疏的分泌囊泡。在所有分泌囊泡群体中，我们在此聚焦于突触样微囊泡（SLMVs）。我们阐述了使用EWi的TIRF显微镜如何有助于在单囊泡水平研究SLMVs的胞吐作用和再循环，并且当与落射荧光照明（EPIi）结合时，能够在全细胞水平提供有关胞吐作用、内吞作用和再酸化动力学的详细信息。
