Zhao Rui, Rueda David
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.
Methods. 2009 Oct;49(2):112-7. doi: 10.1016/j.ymeth.2009.04.017. Epub 2009 May 4.
Single-molecule fluorescence resonance energy transfer (smFRET) microscopy has become an increasingly popular tool to study the structural dynamics of RNA molecules. It reveals, in real time, the structural dynamics of these molecules that would be otherwise hidden in ensemble-averaged measurements. Here we present a detailed protocol for performing smFRET using total internal reflection fluorescence microscopy, including RNA preparation, optical setup, separation of the dual color channels, sample immobilization and data acquisition and analysis.
单分子荧光共振能量转移（smFRET）显微镜已成为研究RNA分子结构动力学越来越受欢迎的工具。它能实时揭示这些分子的结构动力学，而这些动力学在总体平均测量中原本是隐藏的。在这里，我们展示了使用全内反射荧光显微镜进行smFRET的详细方案，包括RNA制备、光学设置、双色通道分离、样品固定以及数据采集和分析。
