Univ Rennes, CNRS, IGDR (Genetics and Development Institute of Rennes), UMR 6290, F-35000 Rennes, France.
Methods Appl Fluoresc. 2020 Feb 20;8(2):024006. doi: 10.1088/2050-6120/ab73f5.
Fluorescence Lifetime Imaging Microscopy (FLIM) is a robust tool to measure Förster Resonance Energy Transfer (FRET) between two fluorescent proteins, mainly when using genetically-encoded FRET biosensors. It is then possible to monitor biological processes such as kinase activity with a good spatiotemporal resolution and accuracy. Therefore, it is of interest to improve this methodology for future high content screening purposes. We here implement a time-gated FLIM microscope that can image and quantify fluorescence lifetime with a higher speed than conventional techniques such as Time-Correlated Single Photon Counting (TCSPC). We then improve our system to perform automatic screen analysis in a 96-well plate format. Moreover, we use a FRET biosensor of AURKA activity, a mitotic kinase involved in several epithelial cancers. Our results show that our system is suitable to measure FRET within our biosensor paving the way to the screening of novel compounds, potentially allowing to find new inhibitors of AURKA activity.
荧光寿命成像显微镜（FLIM）是一种强大的工具，可用于测量两种荧光蛋白之间的Förster 共振能量转移（FRET），主要用于使用遗传编码的 FRET 生物传感器时。然后，可以以良好的时空分辨率和准确性来监测激酶活性等生物过程。因此，为了未来的高通量筛选目的，改进这种方法很有意义。我们在这里实现了一种时间门控 FLIM 显微镜，它可以比传统技术（如时间相关单光子计数（TCSPC））更快地成像和量化荧光寿命。然后，我们改进了系统，以便在 96 孔板格式中进行自动屏幕分析。此外，我们使用了一种 AURKA 活性的 FRET 生物传感器，AURKA 是一种参与多种上皮癌的有丝分裂激酶。我们的结果表明，我们的系统适用于测量我们的生物传感器内的 FRET，为新型化合物的筛选铺平了道路，有可能找到 AURKA 活性的新抑制剂。
