Tsien Roger Y
Cold Spring Harb Protoc. 2009 Jul;2009(7):pdb.top57. doi: 10.1101/pdb.top57.
One of the major new trends in the design of indicators for optically imaging biochemical and physiological functions of living cells has been the exploitation of fluorescence resonance energy transfer (FRET). FRET is a well-known spectroscopic technique for monitoring changes in the proximity and mutual orientation of pairs of chromophores. It has long been used in biochemistry and cell biology to assess distances and orientations between specific labeling sites within a single macromolecule or between two separate molecules. More recently, macromolecules or molecular pairs have been engineered to change their FRET in response to biochemical and physiological signals such as membrane potential, cyclic AMP (cAMP), protease activity, free Ca(2+) and Ca(2+)-calmodulin (CaM) concentrations, protein-protein heterodimerization, phosphorylation, and reporter-gene expression. Because FRET is general, nondestructive, and easily imaged, it has proven to be one of the most versatile spectroscopic readouts available to the designer of new probes. FRET is particularly amenable to emission ratioing, which is more reliably quantifiable than single-wavelength monitoring and better suited than excitation ratioing to high-speed and laser-excited imaging. This article summarizes the photophysical principles of FRET and the types of indicators used.
用于光学成像活细胞生化和生理功能的指标设计中的一个主要新趋势是对荧光共振能量转移（FRET）的利用。FRET是一种用于监测发色团对之间的接近度和相互取向变化的著名光谱技术。长期以来，它一直被用于生物化学和细胞生物学中，以评估单个大分子内特定标记位点之间或两个单独分子之间的距离和取向。最近，已经设计出大分子或分子对，使其能够响应诸如膜电位、环磷酸腺苷（cAMP）、蛋白酶活性、游离Ca(2+)和Ca(2+)-钙调蛋白（CaM）浓度、蛋白质-蛋白质异源二聚化、磷酸化以及报告基因表达等生化和生理信号来改变其FRET。由于FRET具有通用性、非破坏性且易于成像，它已被证明是新探针设计者可用的最通用的光谱读数之一。FRET特别适合发射比率测量，发射比率测量比单波长监测更可靠地可量化，并且比激发比率测量更适合高速和激光激发成像。本文总结了FRET的光物理原理以及所使用的指标类型。
