单分子全内反射荧光显微镜
Single-Molecule Total Internal Reflection Fluorescence Microscopy.
作者信息
Kudalkar Emily M, Davis Trisha N, Asbury Charles L
机构信息
Department of Biochemistry, University of Washington, Seattle, Washington 98195;
Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195.
出版信息
Cold Spring Harb Protoc. 2016 May 2;2016(5):pdb.top077800. doi: 10.1101/pdb.top077800.
The advent of total internal reflection fluorescence (TIRF) microscopy has permitted visualization of biological events on an unprecedented scale: the single-molecule level. Using TIRF, it is now possible to view complex biological interactions such as cargo transport by a single molecular motor or DNA replication in real time. TIRF allows for visualization of single molecules by eliminating out-of-focus fluorescence and enhancing the signal-to-noise ratio. TIRF has been instrumental for studying in vitro interactions and has also been successfully implemented in live-cell imaging. Visualization of cytoskeletal structures and dynamics at the plasma membrane, such as endocytosis, exocytosis, and adhesion, has become much clearer using TIRF microscopy. Thanks to recent advances in optics and commercial availability, TIRF microscopy is becoming an increasingly popular and user-friendly technique. In this introduction, we describe the fundamental properties of TIRF microscopy and the advantages of using TIRF for single-molecule investigation.
全内反射荧光（TIRF）显微镜的出现使人们能够以前所未有的规模——单分子水平观察生物事件。使用TIRF，现在可以实时观察复杂的生物相互作用，如单个分子马达的货物运输或DNA复制。TIRF通过消除离焦荧光并提高信噪比，实现了单分子的可视化。TIRF对研究体外相互作用起到了重要作用，并且也已成功应用于活细胞成像。利用TIRF显微镜，质膜上细胞骨架结构和动态变化（如内吞作用、外排作用和黏附）的可视化变得更加清晰。由于光学技术的最新进展以及商业可得性，TIRF显微镜正成为一种越来越受欢迎且用户友好的技术。在本引言中，我们描述了TIRF显微镜的基本特性以及使用TIRF进行单分子研究的优势。
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