Department of Biomedical Engineering, Rutgers University, Piscataway, New Jersey, USA.
School of Life Sciences Engineering, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland.
Cytometry A. 2021 Apr;99(4):407-416. doi: 10.1002/cyto.a.24194. Epub 2020 Aug 21.
Fӧrster (or fluorescence) resonance energy transfer (FRET) is a quantifiable energy transfer in which a donor fluorophore nonradiatively transfers its excitation energy to an acceptor fluorophore. A change in FRET efficiency indicates a change of proximity and environment of these fluorophores, which enables the study of intermolecular interactions. Measurement of FRET efficiency using the sensitized emission method requires a donor-acceptor calibrated system. One of these calibration factors named the G factor, which depends on instrument parameters related to the donor and acceptor measurement channels and on the fluorophores quantum efficiencies, can be determined in several different ways and allows for conversion of the raw donor and acceptor emission signals to FRET efficiency. However, the calculated value of the G factor from experimental data can fluctuate significantly depending on the chosen experimental method and the size of the sample. In this technical note, we extend the results of Gates et al. (Cytometry Part A 95A (2018) 201-213) by refining the calibration method used for calibration of FRET from image pixel data. Instead of using the pixel histograms of two constructs with high and low FRET efficiency to determine the G factor, we use pixel histogram data from one construct of known efficiency. We validate this method by determining the G factor with the same constructs developed and used by Gates et al. and comparing the results from the two approaches. While the two approaches are equivalent theoretically, we demonstrate that the use of a single construct with known efficiency provides a more precise experimental measurement of the G factor that can be attained by collecting a smaller number of images. © 2020 International Society for Advancement of Cytometry.
Förster（或荧光）共振能量转移（FRET）是一种可量化的能量转移，其中供体荧光团非辐射地将其激发能转移到受体荧光团。FRET 效率的变化表明这些荧光团的接近度和环境发生了变化，这使得能够研究分子间相互作用。使用敏化发射法测量 FRET 效率需要一个供体-受体校准系统。这些校准因子之一称为 G 因子，它取决于与供体和受体测量通道相关的仪器参数以及荧光团的量子效率，可以通过几种不同的方式确定，并允许将原始供体和受体发射信号转换为 FRET 效率。然而，从实验数据计算出的 G 因子值可能会根据所选的实验方法和样品的大小而显著波动。在本技术说明中，我们通过改进用于从图像像素数据校准 FRET 的校准方法扩展了 Gates 等人的结果（《细胞计量学 Part A 95A（2018）201-213》）。我们不是使用具有高低 FRET 效率的两个结构的像素直方图来确定 G 因子，而是使用具有已知效率的一个结构的像素直方图数据。我们通过使用与 Gates 等人开发和使用的相同结构来确定 G 因子来验证这种方法，并比较两种方法的结果。虽然从理论上讲，这两种方法是等效的，但我们证明，使用具有已知效率的单个结构可以通过收集较少数量的图像来更精确地测量 G 因子。
