PIE-FLIM Measurements of Two Different FRET-Based Biosensor Activities in the Same Living Cells.
PIE-FLIM Measurements of Two Different FRET-Based Biosensor Activities in the Same Living Cells.
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PIE-FLIM 测量同一活细胞中两种不同的基于 FRET 的生物传感器活性。
DOI：
10.1016/j.bpj.2020.03.003
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发表时间：
2020
影响因子：
3.4
通讯作者：
Day,RichardN
中科院分区：
文献类型：
--
作者：
Reissaus,ChristopherA;Day,KathleenH;Mirmira,RaghavendraG;Dunn,KennethW;Pavalko,FredrickM;Day,RichardN
我们报告使用脉冲交错激发(PIE)-荧光寿命成像显微镜（FLIM）测量两个不同的生物传感器探针的活性同时在单个活细胞。许多基因编码的生物传感器依赖于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。定量FRET的最稳健的方法之一是测量的变化，在供体荧光团的荧光寿命使用薄膜。对活细胞中复杂信号网络的研究需要同时跟踪多个细胞事件的能力。在这里，我们展示了PIE-FLIM如何分离和量化来自不同的基于fret的生物传感器的信号，同时测量组织中同一活细胞中两种细胞信号通路的活性变化。这里描述的成像系统使用可选择的激光波长和同步检测门控，可以为每个FRET对量身定制和优化。原理证明研究显示同时测量胞质钙和蛋白激酶A活性，但PIE-FLIM方法广泛适用于其他信号通路。
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.