Single-molecule FRET with total internal reflection microscopy.
Single-molecule FRET with total internal reflection microscopy.
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DOI：
10.1101/pdb.top072058
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发表时间：
2012-12-01
影响因子：
--
通讯作者：
Ha, Taekjip
中科院分区：
文献类型：
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作者：
Joo, Chirlmin;Ha, Taekjip
单分子荧光检测是一种无需时间和群体平均的研究生物事件的有效方法。福斯特（荧光）共振能量转移（FRET）是一种光谱技术，其中从供体分子到受体分子的能量转移效率用于确定30-80 A范围内的分子之间的距离。生物分子的结构变化或两个相互作用分子之间的相对运动可以通过FRET的变化来检测。本文主要关注基于全内反射（TIR）显微镜的smFRET。它开始讨论染料的选择和标记的核酸和蛋白质。随后是关于表面准备和数据采集的信息。然后介绍了各种数据分析方法，以及有关设置TIR显微镜的信息，包括物镜和棱镜类型。
Single-molecule (sm) fluorescence detection is a powerful method for studying biological events without time and population averaging. Forster (fluorescence) resonance energy transfer (FRET) is a spectroscopic technique in which the efficiency of energy transfer from donor to acceptor molecules is used to determine distances between molecules in the 30-80 A range. Structural changes in biological molecules or relative motion between two interacting molecules can be detected by a change in FRET. This article focuses primarily on smFRET based on total internal reflection (TIR) microscopy. It begins with discussions of dye choice and labeling of nucleic acids and proteins. These are followed by information on surface preparation and data acquisition. Various methods of data analysis are then presented, as is information on setting up TIR microscopy, both the objective and the prism types.