本文发展了一种针对于固定FRET质粒的单波长激发的E-FRET方法(SDW-E-FRET)。相比于E-FRET方法, SDW-E-FRET只需要测量供体激发时供体通道的荧光强度(IDD)和FRET通道的荧光强度(IDA), 因此该方法可以实现活细胞的快速定量FRET成像。结合双通道荧光显微成像系统, 该方法无需任何机械切换, 定量FRET成像的速度只取决于CCD相机的成像速度, 因而特别适合活细胞实时动态定量FRET成像。在本研究小组发展的双通道荧光显微镜平台上, 应用SDW-E-FRET方法测量了C5V和C17V的FRET效率, 得到了与其它方法测量的一致的结果。

We developed a single-wavelength excitation-based quantitative E-FRET measurement method (SDW-E-FRET). This method only needs to collect fluorescence intensities of donor channel (IDD) and FRET channel (IDA) under donor excitation, and can realize rapid FRET imaging in living cells. Combining the dual-channel fluorescence microscopic imaging system, SDW-E-FRET method does not need any mechanical switch, and the speed of quantitative SDW-E-FRET imaging only depends on the speed of CCD camera. Therefore, SDW-E-FRET is applicable to mapping the dynamics of intracellular process in live cells in real time. We performed quantitative SDW-E-FRET measurement on our dual-channel fluorescence microscope to measure the FRET efficiency of C5V and C17V in living cells, and obtained consistent results with those measured by other methods.