Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex
Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex
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DOI：
10.1111/j.1365-313x.2007.03306.x
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发表时间：
2008-01-01
期刊：
影响因子：
7.2
通讯作者：
Bednarek, Sebastian Y.
中科院分区：
文献类型：
--
作者：
Konopka, Catherine A.;Bednarek, Sebastian Y.
荧光标记融合蛋白的活细胞显微镜成像是细胞生物学家的重要工具。全内反射荧光显微镜（TIRFM）已加入共聚焦显微镜作为一个补充系统，在哺乳动物和酵母系统的细胞表面蛋白质动态成像，因为它的高时间和空间分辨率。在这里，我们提出了一种替代TIRFM，称为可变角度落射荧光显微镜（VAEM），用于可视化蛋白质动态或附近的质膜的植物表皮细胞和根毛在整个，完整的幼苗，提供高信号，低背景和近实时成像。VAEM使用高度倾斜的亚临界入射角来减少背景荧光团激发。我们讨论了VAEM的效用和优势，研究皮质细胞骨架和膜蛋白的荧光融合标记蛋白成像。我们相信，VAEM的应用将是植物细胞生物学家的一个宝贵的成像工具。
Live-cell microscopy imaging of fluorescent-tagged fusion proteins is an essential tool for cell biologists. Total internal reflection fluorescence microscopy (TIRFM) has joined confocal microscopy as a complementary system for the imaging of cell surface protein dynamics in mammalian and yeast systems because of its high temporal and spatial resolution. Here we present an alternative to TIRFM, termed variable-angle epifluorescence microscopy (VAEM), for the visualization of protein dynamics at or near the plasma membrane of plant epidermal cells and root hairs in whole, intact seedlings that provides high-signal, low-background and near real-time imaging. VAEM uses highly oblique subcritical incident angles to decrease background fluorophore excitation. We discuss the utilities and advantages of VAEM for imaging of fluorescent fusion-tagged marker proteins in studying cortical cytoskeletal and membrane proteins. We believe that the application of VAEM will be an invaluable imaging tool for plant cell biologists.