Imaging FRET standards by steady-state fluorescence and lifetime methods
Imaging FRET standards by steady-state fluorescence and lifetime methods
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DOI：
10.1002/jemt.20509
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发表时间：
2007-12-01
影响因子：
2.5
通讯作者：
Llopis, Juan
中科院分区：
文献类型：
--
作者：
Domingo, Beatriz;Sabariegos, Rosario;Llopis, Juan
荧光蛋白标记分子间的成像荧光共振能量转移(FRET)是研究生理细胞环境中离子、配体和分子相互作用变化的有力工具。不同的方法使用稳态荧光特性或寿命来量化FRET速率。此外，有些提供了绝对的FRET效率，而另一些只是一个受实际设置和使用的仪器影响很大的相对指数，这使得对给定FRET速率的解释非常困难。在使用这些技术的实验室中使用和交换FRET标准将有助于克服这一缺点。我们报告了不同FRET效率的FRET标准探针的构建和系统评价。分子内FRET的标准是由短链或更大的蛋白间隔物连接的维多利亚绿色荧光蛋白的青色和黄色变体(ECFP和柠檬碱)的蛋白质融合，或标记有四半胱氨酸基序并标记有双砷荧光色素的ECFP。同时采用分子间FRET阴性对照和阳性对照。我们将这些FRET标准与多达四种FRET定量方法进行了比较：受体与供体辐射的比率、受体光漂白后的供体强度恢复、原始图像光谱分解后的敏化发射和荧光寿命成像(FLIM)。后者是通过频域设置获得的，能够在不到一秒的时间内提供高质量的寿命图像，因此非常适合活体细胞研究。无论使用哪种方法，标准的FRET率或指数都是一致的。对于CFP-四半胱氨酸/闪光对，由CFP猝灭计算的速度比用Flim计算的速度快。
Imaging fluorescence resonance energy transfer (FRET) between molecules labeled with fluorescent proteins is emerging as a powerful tool to study changes in ions, ligands, and molecular interactions in their physiological cellular environment. Different methods use either steady-state fluorescence properties or lifetime to quantify the FRET rate. In addition, some provide the absolute FRET efficiency whereas others are simply a relative index very much influenced by the actual settings and instrumentation used, which makes the interpretation of a given FRET rate very difficult. The use and exchange of FRET standards in laboratories using these techniques would help to overcome this drawback. We report here the construction and systematic evaluation of FRET standard probes of varying FRET efficiencies. The standards for intramolecular FRET were protein fusions of the cyan and yellow variants of A. victoria green fluorescent protein (ECFP and citrine) joined by short linkers or larger protein spacers, or ECFP tagged with a tetracysteine motif and labeled with the biarsenical fluorochrome, FlAsH. Negative and positive controls of intermolecular FRET were also used. We compared these FRET standards with up to four FRET quantification methods: ratioing of acceptor to donor emission, donor intensity recovery upon acceptor photobleach, sensitized emission after spectral unmixing of raw images, and fluorescence lifetime imaging (FLIM). The latter was obtained with a frequency-domain setup able to provide high quality lifetime images in less than a second, and is thus very well suited for live cell studies. The FRET rates or indexes of the standards Were in good agreement regardless of the method used. For the CFP-tetraCys/FlAsH pair, the rate calculated from CFP quenching was faster than that obtained by FLIM.