Brooks F Phil, Gong Daozheng, Davis Hunter C, Park Pojeong, Qi Yitong, Cohen Adam E
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
Sci Adv. 2025 Jan 10;11(2):eadp5763. doi: 10.1126/sciadv.adp5763. Epub 2025 Jan 8.
Microbial rhodopsin-derived genetically encoded voltage indicators (GEVIs) are powerful tools for mapping bioelectrical dynamics in cell culture and in live animals. Förster resonance energy transfer (FRET)-opsin GEVIs use voltage-dependent quenching of an attached fluorophore, achieving high brightness, speed, and voltage sensitivity. However, the voltage sensitivity of most FRET-opsin GEVIs has been reported to decrease or vanish under two-photon (2P) excitation. Here, we investigated the photophysics of the FRET-opsin GEVIs Voltron1 and Voltron2. We found that the previously reported negative-going voltage sensitivities of both GEVIs came from photocycle intermediates, not from the opsin ground states. The voltage sensitivities of both GEVIs were nonlinear functions of illumination intensity; for Voltron1, the sensitivity reversed the sign under low-intensity illumination. Using photocycle-optimized 2P illumination protocols, we demonstrate 2P voltage imaging with Voltron2 in the barrel cortex of a live mouse. These results open the door to high-speed 2P voltage imaging of FRET-opsin GEVIs in vivo.
源自微生物视紫红质的基因编码电压指示剂（GEVIs）是用于绘制细胞培养物和活体动物生物电动力学的强大工具。荧光共振能量转移（FRET）-视蛋白GEVIs利用附着荧光团的电压依赖性猝灭，实现了高亮度、速度和电压敏感性。然而，据报道，大多数FRET-视蛋白GEVIs的电压敏感性在双光子（2P）激发下会降低或消失。在这里，我们研究了FRET-视蛋白GEVIs Voltron1和Voltron2的光物理性质。我们发现，先前报道的两种GEVIs的负向电压敏感性来自光循环中间体，而非视蛋白基态。两种GEVIs的电压敏感性都是光照强度的非线性函数；对于Voltron1，在低强度光照下敏感性的符号会反转。使用光循环优化的2P照明方案，我们在活体小鼠的桶状皮层中展示了用Voltron2进行的2P电压成像。这些结果为体内FRET-视蛋白GEVIs的高速2P电压成像打开了大门。
