Two-Photon Lifetime Imaging of Voltage Indicating Proteins as a Probe of Absolute Membrane Voltage
Two-Photon Lifetime Imaging of Voltage Indicating Proteins as a Probe of Absolute Membrane Voltage
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DOI：
10.1016/j.bpj.2015.07.038
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发表时间：
2015-09-01
影响因子：
3.4
通讯作者：
Cohen, Adam E.
中科院分区：
文献类型：
--
作者：
Brinks, Daan;Klein, Aaron J.;Cohen, Adam E.
遗传编码电压指示器（GEVI）可以在空间和时间上以高分辨率报告细胞电生理学。双光子（2 P）荧光已经被探索作为对组织中的电压成像的手段。在这里，我们使用的2 P电子激发态寿命探测绝对膜电压的方式是不敏感的蛋白质表达水平，照明强度，或光子检测效率。首先，我们测试了几个GEVI的2 P亮度，响应速度和电压灵敏度。ASAP 1和先前描述的柠檬酸-Arch电致变色福斯特共振能量转移传感器（称为CAESR）显示出最好的特性。然后，我们的特点是电压钳条件下的ASAP 1，CAESR，和弧光电压依赖性的寿命。ASAP 1和CAESR显示出电压依赖性寿命，而ArcLight则没有。这些结果建立2 P荧光寿命成像作为测量绝对膜电压的可行手段。我们讨论了在组织中应用的前景和必要的改进。
Genetically encoded voltage indicators (GEVIs) can report cellular electrophysiology with high resolution in space and time. Two-photon (2P) fluorescence has been explored as a means to image voltage in tissue. Here, we used the 2P electronic excited-state lifetime to probe absolute membrane voltage in a manner that is insensitive to the protein expression level, illumination intensity, or photon detection efficiency. First, we tested several GEVIs for 2P brightness, response speed, and voltage sensitivity. ASAP1 and a previously described citrine-Arch electrochromic Forster resonance energy transfer sensor (dubbed CAESR) showed the best characteristics. We then characterized the voltage-dependent lifetime of ASAP1, CAESR, and ArcLight under voltage-clamp conditions. ASAP1 and CAESR showed voltage-dependent lifetimes, whereas ArcLight did not. These results establish 2P fluorescence lifetime imaging as a viable means of measuring absolute membrane voltage. We discuss the prospects and improvements necessary for applications in tissue.