Fluorescence resonance energy transfer-based stoichiometry in living cells
Fluorescence resonance energy transfer-based stoichiometry in living cells
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DOI：
10.1016/s0006-3495(02)75365-4
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发表时间：
2002-12-01
影响因子：
3.4
通讯作者：
Swanson, JA
中科院分区：
文献类型：
--
作者：
Hoppe, A;Christensen, K;Swanson, JA
荧光标记分子之间的荧光共振能量转移(FRET)成像可以测量活细胞内分子间相互作用的时间和位置。现有的微观方法测量FRET的单位是任意的，不能区分FRET的效率和络合物中供体和受体的比例。在这里，我们描述了一种化学计量学方法，该方法使用三个显微荧光图像来测量活细胞中FRET效率、供体和受体的相对浓度以及复合体中供体和受体的比例。FRET化学计量学源于这样的概念，即特定的供体-受体络合物将产生特征的FRET效率，如果测量该效率，可以允许相互作用组分的化学计量区分。第一个方程决定了FRET效率和受体分子与给体形成络合物的比例。第二个方程通过估计由于能量转移而损失的给体荧光来确定给体分子在络合物中的比例。这就不需要受体光漂白来确定总的供体浓度，并允许对同一细胞进行重复测量。第三个方程得到总受体与总给体分子的比率。通过对青色荧光蛋白(CFP)、黄碱和连接的CFP-Citine融合蛋白在溶液中和细胞内的荧光显微测量，验证了该理论和方法。从这些方程中得出的方法可以在图像的每个像素上灵敏、快速和可重复地检测供体-受体-和供体-受体复合体的化学计量比。通过精确成像分子相互作用，FRET化学计量学为细胞内分子网络的定量研究开辟了新的领域。
Imaging of fluorescence resonance energy transfer (FRET) between fluorescently labeled molecules can measure the timing and location of intermolecular interactions inside living cells. Present microscopic methods measure FRET in arbitrary units, and cannot discriminate FRET efficiency and the fractions of donor and acceptor in complex. Here we describe a stoichiometric method that uses three microscopic fluorescence images to measure FRET efficiency, the relative concentrations of donor and acceptor, and the fractions of donor and acceptor in complex in living cells. FRET stoichiometry derives from the concept that specific donor-acceptor complexes will give rise to a characteristic FRET efficiency, which, if measured, can allow stoichiometric discrimination of interacting components. A first equation determines FRET efficiency and the fraction of acceptor molecules in complex with donor. A second equation determines the fraction of donor molecules in complex by estimating the donor fluorescence lost due to energy transfer. This eliminates the need for acceptor photo-bleaching to determine total donor concentrations and allows for repeated measurements from the same cell. A third equation obtains the ratio of total acceptor to total donor molecules. The theory and method were confirmed by microscopic measurements of fluorescence from cyan fluorescent protein (CFP), citrine, and linked CFP-Citrine fusion protein, in solutions and inside cells. Together, the methods derived from these equations allow sensitive, rapid, and repeatable detection of donor-, acceptor-, and donor-acceptor complex stoichiometry at each pixel in an image. By accurately imaging molecular interactions, FRET stoichiometry opens new areas for quantitative study of intracellular molecular networks.