Windgasse Lukas, Grashoff Carsten
Department of Quantitative Cell Biology, Institute of Integrative Cell Biology and Physiology, University of Münster, Münster, Germany.
Methods Mol Biol. 2023;2600:221-237. doi: 10.1007/978-1-0716-2851-5_15.
Genetically encoded Förster Resonance Energy Transfer (FRET)-based tension sensors were developed to enable the quantification of piconewton (pN)-scale forces that act across distinct proteins in living cells and organisms. An important extension of this technology is the multiplexing of tension sensors to monitor several independent FRET probes in parallel. Here we describe how pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) can be implemented to enable the analysis of two co-expressed tension sensor constructs. Our protocol covers all essential steps from biosensor expression and live cell PIE image acquisition to lifetime calculations.
基于基因编码的Förster共振能量转移（FRET）张力传感器已被开发出来，用于量化作用于活细胞和生物体中不同蛋白质的皮牛顿（pN）级别的力。该技术的一个重要扩展是张力传感器的多路复用，以便并行监测多个独立的FRET探针。在这里，我们描述了如何实施脉冲交错激发（PIE）-荧光寿命成像显微镜（FLIM），以分析两种共表达的张力传感器构建体。我们的方案涵盖了从生物传感器表达、活细胞PIE图像采集到寿命计算的所有基本步骤。
