由于天然克服光谱串扰的能力以及高灵敏和无损伤的特性,基于光谱分离的荧光共振能量转移(FRET)定量检测(spFRET)方法被公认为是最有应用潜力的活细胞定量FRET检测技术。首先简要介绍FRET定量检测方法以及国内外的相关研究进展;其次重点介绍基于发射光谱线性分离(Em-unmixing)和基于激发发射光谱线性分离(ExEm-unmixing)的两种定量FRET检测技术的原理、发展进程,并比较了这两种检测技术的稳健性;最后对这两种spFRET技术在活细胞FRET应用中的潜在优势进行展望。

Owing to its inherent ability to overcome the spectral crosstalk, high sensitivity, and non-destructivity characteristic, fluorescence resonance energy transfer (FRET) quantitative measurement (spFRET) method based on spectral unmixing has been generally regarded as the most promising approach of live-cell FRET measurement. This paper first briefly introduced the quantitative FRET measurement method and the related research advances on FRET technology. Second, the principle, development process, and robustness of spFRET based on linear separation of emission spectra (Em-unmixing) and linear separation of excitation emission spectra (ExEm-unmixing), respectively, were introduced. Finally, the potential advantages of the two spFRET technologies in live-cell FRET applications was also provided and discussed.