Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN, USA.
Bioessays. 2012 May;34(5):341-50. doi: 10.1002/bies.201100098. Epub 2012 Mar 7.
The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for fluorescence (or Förster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, the potential for photoconversion artifacts and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs.
新型荧光蛋白（FPs）的发现和工程改造源于不同的生物体，产生了具有特殊活细胞成像特性的荧光团。特别是，为荧光（或福斯特）共振能量转移（FRET）显微镜开发的荧光蛋白为监测活细胞内动态蛋白质相互作用提供了重要工具。对 FRET 显微镜的日益关注推动了许多不同的 FRET 测量方法的发展。然而，由于高荧光背景、潜在的光转化伪影以及该技术相对较低的动态范围等因素，FRET 测量的解释变得复杂。在这里，我们描述了 FRET 显微镜中常用的四种方法的优缺点。然后，我们讨论了不同 FRET 方法中 FP 的选择，确定了最适合 FRET 显微镜的 FP 候选物。最近在扩展 FP 调色板方面的成功为探索新的 FRET 对提供了机会。
