Evanescent-wave microscopy:: a new tool to gain insight into the control of transmitter release
Evanescent-wave microscopy:: a new tool to gain insight into the control of transmitter release
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DOI：
10.1098/rstb.1999.0382
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发表时间：
1999-02-28
影响因子：
6.3
通讯作者：
Stühmer, W
中科院分区：
文献类型：
--
作者：
Oheim, M;Loerke, D;Stühmer, W
使用倏逝波激发来观察牛肾上腺嗜铬细胞质膜附近光学切片中单个荧光标记的囊泡。对标准的正置显微镜进行了修改，以适应用于将超临界角度的激光束引导到细胞生长的玻璃-水界面上的光学器件。落射照明图像显得模糊且无结构，而倏逝波激发则将吖啶橙标记的囊泡突出为单独的精确点。从时间分辨图像堆栈中获得单个囊泡的三维 (3D) 轨迹，并用于根据囊泡的平均荧光 F 和迁移率来表征囊泡，此处表示为 3D 扩散系数 D-(3)。基于单囊泡分析，鉴定了两组囊泡。通过重复或维持细胞外 [K+] 升高的膜去极化来刺激胞吐作用之前和之后，研究了这些状态之间的转变。结果被解释为融合步骤之前先前表征的囊泡池之间的顺序转变。观察到的：囊泡接近其对接位点并不能用自由扩散来解释：大多数囊泡单向移动，就好像定向到它们在质膜上的结合位点一样。膜上的囊泡迁移率较低，因此对接和融合的位点非常接近。足迹区域的边缘区域和中心区域都是囊泡贩运密集的场所。
Evanescent-wave excitation was used to visualize individual fluorescently labelled vesicles in an optical slice near the plasma membrane of bovine adrenal chromaffin cells. A standard upright microscope was modified to accommodate the optics used for directing a laser beam under a supracritical angle on to the glass-water interface on top of which the cells are grown. Whereas epi-illumination images appeared blurred and structureless, evanescent-wave excitation highlighted acridine orange-labelled vesicles as individual pinpoints. Three-dimensional (3D) trajectories of individual vesicles were obtained from time-resolved image stacks and used to characterize vesicles in terms of their average fluorescence F and mobility, expressed here as the 3D diffusion coefficient D-(3). Based on the single-vesicle analysis, two groups of vesicles were identified. Transitions between these states were studied before and after stimulation of exocytosis by repetitive or maintained membrane depolarizations by elevated extracellular [K+]. Findings were interpreted as sequential transitions between the previously characterized pools of vesicles preceding the fusion step. The observed: approach of vesicles to their docking sites was not explained in terms of free diffusion: most vesicles moved unidirectionally as if directed to their binding sites at the plasma membrane. Vesicle mobility at the membrane was low, such that the sites of docking and fusion were in close vicinity. Both the rim region and confined areas in the centre of the footprint region were the site of intense vesicle trafficking.