We report the use of pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) to measure the activities of two different biosensor probes simultaneously in single living cells. Many genetically encoded biosensors rely on the measurement of Förster resonance energy transfer (FRET) to detect changes in biosensor conformation that accompany the targeted cell signaling event. One of the most robust ways of quantifying FRET is to measure changes in the fluorescence lifetime of the donor fluorophore using FLIM. The study of complex signaling networks in living cells demands the ability to track more than one of these cellular events at the same time. Here, we demonstrate how PIE-FLIM can separate and quantify the signals from different FRET-based biosensors to simultaneously measure changes in the activity of two cell signaling pathways in the same living cells in tissues. The imaging system described here uses selectable laser wavelengths and synchronized detection gating that can be tailored and optimized for each FRET pair. Proof-of-principle studies showing simultaneous measurement of cytosolic calcium and protein kinase A activity are shown, but the PIE-FLIM approach is broadly applicable to other signaling pathways.

中文翻译：

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同一活细胞中两种不同的基于 FRET 的生物传感器活性的 PIE-FLIM 测量

我们报告了使用脉冲交错激发 (PIE)-荧光寿命成像显微镜 (FLIM) 在单个活细胞中同时测量两种不同生物传感器探针的活动。许多基因编码的生物传感器依靠 Förster 共振能量转移 (FRET) 的测量来检测伴随目标细胞信号事件的生物传感器构象的变化。量化 FRET 的最可靠方法之一是使用 FLIM 测量供体荧光团的荧光寿命变化。活细胞中复杂信号网络的研究需要能够同时跟踪多个细胞事件。这里，我们展示了 PIE-FLIM 如何分离和量化来自不同基于 FRET 的生物传感器的信号，以同时测量组织中相同活细胞中两种细胞信号通路的活性变化。此处描述的成像系统使用可选择的激光波长和同步检测门控，可以针对每个 FRET 对进行定制和优化。显示了同时测量细胞溶质钙和蛋白激酶 A 活性的原理验证研究，但 PIE-FLIM 方法广泛适用于其他信号通路。