荧光共振能量转移 (FRET) 生物传感器已被证明是细胞生物学中不可或缺的工具，更具体地说，是 G 蛋白信号传导研究中不可或缺的工具。测量生物传感器的激活状态或 FRET 状态的最佳方法通常是荧光寿命成像显微镜 (FLIM)，因为它消除了基于荧光强度的方法固有的许多缺点，并且易于定量。尽管潜力巨大，但仍缺乏可靠的 FLIM-FRET 生物传感器，并且之前报道的数据处理和分析工作流程面临重现性挑战。在这里，我们在活的原代小鼠胰腺导管腺癌细胞中建立了一个系统，我们可以通过溶血磷脂酸受体（LPAR）使用 2 光子时间相关的单光子计数来检测 mNeonGreen-Gαi3-mCherry-Gγ2 生物传感器的激活（TCSPC）FLIM。这种组合为常用的 mTurquoise2-mVenus G 蛋白生物传感器提供了更好的信号。该系统有潜力作为药物筛选平台，或回答 G 蛋白信号传导领域的基本细胞生物学问题。

Fluorescence resonance energy transfer (FRET) biosensors have proven to be an indispensable tool in cell biology and, more specifically, in the study of G-protein signalling. The best method of measuring the activation status or FRET state of a biosensor is often fluorescence lifetime imaging microscopy (FLIM), as it does away with many disadvantages inherent to fluorescence intensity-based methods and is easily quantitated. Despite the significant potential, there is a lack of reliable FLIM-FRET biosensors, and the data processing and analysis workflows reported previously face reproducibility challenges. Here, we established a system in live primary mouse pancreatic ductal adenocarcinoma cells, where we can detect the activation of an mNeonGreen-Gαi3-mCherry-Gγ2 biosensor through the lysophosphatidic acid receptor (LPAR) with 2-photon time-correlated single-photon counting (TCSPC) FLIM. This combination gave a superior signal to the commonly used mTurquoise2-mVenus G-protein biosensor. This system has potential as a platform for drug screening, or to answer basic cell biology questions in the field of G-protein signalling.