Imaging of dynamic secretory vesicles in living pollen tubes of Picea meyeri using evanescent wave microscopy
Imaging of dynamic secretory vesicles in living pollen tubes of Picea meyeri using evanescent wave microscopy
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使用倏逝波显微镜对云杉活花粉管中的动态分泌囊泡进行成像
DOI：
10.1104/pp.106.080168
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发表时间：
2006-08-01
期刊：
影响因子：
7.4
通讯作者：
Lin, Jinxing
中科院分区：
文献类型：
--
作者：
Wang, Xiaohua;Teng, Yan;Lin, Jinxing
使用倏逝波激发来可视化靠近云杉花粉管质膜的光学切片中的单个 FM4-64 标记的分泌囊泡。对标准正置显微镜进行了修改，以适应用于以可变角度引导激光束的光学器件。在倏逝波显微镜或全内反射荧光显微镜下，位于表面附近的荧光团被倏逝波激发，倏逝波随着距界面的距离呈指数衰减。通过改变激光束的入射角产生穿透深度为 60 至 400 nm 的倏逝波。使用单个荧光标记囊泡的延时倏逝波成像，通过质膜下方约 300 nm 的光学切片进行囊泡运输的动力学分析。从所得的时间分辨图像堆栈中获得单个囊泡的二维轨迹，并用于根据囊泡的平均荧光和迁移率来表征囊泡，此处表示为二维扩散系数 D-2。还计算了囊泡运动的速度和方向、框架到框架的位移以及囊泡轨迹。据我们所知，对单个囊泡的分析首次揭示了存在两种类型的运动，并且活花粉管中的囊泡表现出复杂的行为和振荡，与之前研究中报告的简单布朗运动不同。此外，肌动蛋白细胞骨架的破坏对囊泡迁移性的影响比微管的破坏更显着，表明肌动蛋白细胞骨架在囊泡迁移性中起主要作用。
Evanescent wave excitation was used to visualize individual, FM4-64-labeled secretory vesicles in an optical slice proximal to the plasma membrane of Picea meyeri pollen tubes. A standard upright microscope was modified to accommodate the optics used to direct a laser beam at a variable angle. Under evanescent wave microscopy or total internal reflection fluorescence microscopy, fluorophores localized near the surface were excited with evanescent waves, which decay exponentially with distance from the interface. Evanescent waves with penetration depths of 60 to 400 nm were generated by varying the angle of incidence of the laser beam. Kinetic analysis of vesicle trafficking was made through an approximately 300-nm optical section beneath the plasma membrane using time-lapse evanescent wave imaging of individual fluorescently labeled vesicles. Two-dimensional trajectories of individual vesicles were obtained from the resulting time-resolved image stacks and were used to characterize the vesicles in terms of their average fluorescence and mobility, expressed here as the two-dimensional diffusion coefficient D-2. The velocity and direction of vesicle motions, frame-to-frame displacement, and vesicle trajectories were also calculated. Analysis of individual vesicles revealed for the first time, to our knowledge, that two types of motion are present, and that vesicles in living pollen tubes exhibit complicated behaviors and oscillations that differ from the simple Brownian motion reported in previous investigations. Furthermore, disruption of the actin cytoskeleton had a much more pronounced effect on vesicle mobility than did disruption of the microtubules, suggesting that actin cytoskeleton plays a primary role in vesicle mobility.