Photophysics-informed two-photon voltage imaging using FRET-opsin voltage indicators

• DOI: 10.1126/sciadv.adp5763
• Publication Date: 2025-01-08T18:59:23Z
• Authors: F. Phil Brooks; Daozheng Gong; Hunter C. Davis; Pojeong Park; Yitong Qi; Adam E. Cohen
• Keywords: Opsin; Two-photon excitation microscopy; Fluorescence-lifetime imaging microscopy; Voltage-sensitive dye; Biophotonics

Microbial rhodopsin–derived genetically encoded voltage indicators (GEVIs) are powerful tools for mapping bioelectrical dynamics in cell culture and live animals. Förster resonance energy transfer (FRET)–opsin GEVIs use voltage-dependent quenching of an attached fluorophore, achieving high brightness, speed, sensitivity. However, the sensitivity most FRET-opsin has been reported to decrease or vanish under two-photon (2P) excitation. Here, we investigated photophysics Voltron1 Voltron2. We found that previously negative-going sensitivities both came from photocycle intermediates, not opsin ground states. The were nonlinear functions illumination intensity; Voltron1, reversed sign low-intensity illumination. Using photocycle-optimized 2P protocols, demonstrate imaging with Voltron2 barrel cortex a mouse. These results open door high-speed vivo.