satFRET: estimation of Forster resonance energy transfer by acceptor saturation
satFRET: estimation of Forster resonance energy transfer by acceptor saturation
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DOI：
10.1007/s00249-008-0361-5
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发表时间：
2008-11-01
影响因子：
2
通讯作者：
Heintzmann, Rainer
中科院分区：
文献类型：
--
作者：
Beutler, Martin;Makrogianneli, Konstantina;Heintzmann, Rainer
我们从理论和实验上证明了福斯特共振能量转移（FRET）的受体分子的激发态的直接和系统的饱和的量化。用于FRET估计的该版本的受体耗尽方法（表示为“satFRET”）是可逆的并且适合于时间分辨测量。利用施主和受主分子态微分方程组的稳态解对该技术进行了理论研究。模型中考虑了测量过程中受体光漂白的影响。实验验证是用不同化学计量的FRET对Alexa 546-Alexa 633装载在颗粒上，并在共聚焦显微镜中测量。通过激发态饱和的能量转移效率的估计进行了比较，通过测量敏化发射和受体光漂白。结果导致一个协议，允许时间分辨FRET测量固定和活细胞在传统的共聚焦显微镜。将该程序应用于含有青色荧光蛋白和黄色荧光蛋白对的固定的中国仓鼠卵巢细胞。该技术的时间分辨率在活T细胞活化试验中得到证实，该试验比较了使用遗传编码的绿色和红色荧光蛋白生物传感器测量的用于GTP/GDP周转的FRET效率与通过固定细胞的受体光漂白测量的FRET效率。
We demonstrate theoretically and experimentally the quantification of Forster resonance energy transfer (FRET) by direct and systematic saturation of the excited state of acceptor molecules. This version of acceptor depletion methods for FRET estimation, denoted as "satFRET" is reversible and suitable for time-resolved measurements. The technique was investigated theoretically using the steady-state solution of the differential equation system of donor and acceptor molecular states. The influence of acceptor photobleaching during measurement was included in the model. Experimental verification was achieved with the FRET-pair Alexa 546-Alexa 633 loaded on particles in different stoichiometries and measured in a confocal microscope. Estimates of energy transfer efficiency by excited state saturation were compared to those obtained by measurements of sensitised emission and acceptor photobleaching. The results lead to a protocol that allows time-resolved FRET measurements of fixed and living cells on a conventional confocal microscope. This procedure was applied to fixed Chinese hamster ovary cells containing a cyan fluorescent protein and yellow fluorescent protein pair. The time resolution of the technique was demonstrated in a live T cell activation assay comparing the FRET efficiencies measured using a genetically encoded green and red fluorescent protein biosensor for GTP/GDP turnover to those measured by acceptor photobleaching of fixed cells.