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.

中文翻译：

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使用荧光寿命成像显微镜，基于遗传编码的FRET生物传感器自动筛选AURKA活性。

荧光寿命成像显微镜（FLIM）是一种强大的工具，主要用于使用基因编码的FRET生物传感器来测量两种荧光蛋白之间的福斯特共振能量转移（FRET）。然后可以以良好的时空分辨率和准确性来监视诸如激酶活性的生物学过程。因此，为了将来的高含量筛选目的，改进这种方法是令人感兴趣的。我们在这里实现了一种时间门控FLIM显微镜，该显微镜能以比常规技术（例如时间相关单光子计数（TCSPC））更高的速度成像和量化荧光寿命。然后，我们改进我们的系统，以96孔板的形式进行自动筛选分析。此外，我们使用AURKA活动的FRET生物传感器，AURKA活动是一种参与多种上皮癌的有丝分裂激酶。