Simplified Instrument Calibration forWide-FieldFluorescence Resonance Energy Transfer (FRET) Measured by the Sensitized Emission Method
Simplified Instrument Calibration forWide-FieldFluorescence Resonance Energy Transfer (FRET) Measured by the Sensitized Emission Method
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DOI：
10.1002/cyto.a.24194
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发表时间：
2020-08-21
期刊：
影响因子：
3.7
通讯作者：
Boustany, Nada N.
中科院分区：
文献类型：
--
作者：
Menaesse, Audrey;Sumetsky, Daniel;Boustany, Nada N.
福斯特（或荧光）共振能量转移（FRET）是一种可量化的能量转移，其中供体荧光团将其激发能非辐射地转移到受体荧光团。FRET效率的变化表明这些荧光团的接近和环境的变化，这使得研究分子间相互作用成为可能。使用敏化发射法测量FRET效率需要一个供体-受体校准系统。其中一个被称为g因子的校准因子，它取决于与供体和受体测量通道以及荧光团量子效率相关的仪器参数，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，根据实验数据计算出的G因子的值可能会根据所选择的实验方法和样本的大小而有很大的波动。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（Cytometry Part A 95A(2018) 201-213）的结果。我们没有使用具有高和低FRET效率的两个结构的像素直方图来确定g因子，而是使用来自一个已知效率结构的像素直方图数据。我们通过确定与Gates等人开发和使用的相同结构的g因子并比较两种方法的结果来验证该方法。虽然这两种方法在理论上是等效的，但我们证明，使用具有已知效率的单一结构可以通过收集较少数量的图像来获得更精确的g因子实验测量。(c) 2020年国际细胞术进步学会
Forster (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 theGfactor, 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 theGfactor, we use pixel histogram data from one construct of known efficiency. We validate this method by determining theGfactor 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 theGfactor that can be attained by collecting a smaller number of images. (c) 2020 International Society for Advancement of Cytometry