Protein-protein interactions can be measured in live cells, at nanometer scale, using Fluorescence Lifetime Imaging Microscopy (FLIM) enabled Forster Resonance Energy Transfer (FRET). There are growing interests in exploring protein-protein interactions in drug discovery applications. Traditional single point confocal microscopes, however, are slow and unsuited to small molecule screening, especially when combined with FLIM-FRET. We developed a 32 × 32 multiplexed confocal microscope, which employs a single-photon avalanche photodiode array with time gating capabilities for rapid FLIM acquisition. It has been demonstrated that such multiplexing technique can capture a 960 × 960 pixel multi-channel confocal fluorescence lifetime images in less than 1.5 seconds. Binding curves of two Bcl-2 family proteins: Bcl-XL and Bad were generated in live cells imaging experiments. The results show that the small molecule inhibitor A-1131852 is a more effective compound for disrupting Bcl-XL binding to Bad than ABT-263, which demonstrated the feasibility of screening of protein-protein interactions in high density well-plates.

中文翻译：

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专为筛选应用而设计的高度复用共聚焦荧光寿命显微镜

使用荧光寿命成像显微镜 (FLIM) 启用的福斯特共振能量转移 (FRET)，可以在纳米尺度上测量活细胞中的蛋白质-蛋白质相互作用。人们对探索药物发现应用中的蛋白质-蛋白质相互作用越来越感兴趣。然而，传统的单点共聚焦显微镜速度慢且不适合小分子筛选，尤其是与 FLIM-FRET 结合使用时。我们开发了 32 × 32 多路复用共聚焦显微镜，它采用单光子雪崩光电二极管阵列，具有时间门控功能，可快速获取 FLIM。已经证明，这种多路复用技术可以在不到 1.5 秒的时间内捕获 960 × 960 像素的多通道共焦荧光寿命图像。两种 Bcl-2 家族蛋白的结合曲线：Bcl-XL 和 Bad 在活细胞成像实验中产生。结果表明，小分子抑制剂 A-1131852 是一种比 ABT-263 更有效地破坏 Bcl-XL 与 Bad 结合的化合物，这证明了在高密度孔板中筛选蛋白质-蛋白质相互作用的可行性。