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

Sci Adv. 2025 Jan 10;11(2):eadp5763. doi: 10.1126/sciadv.adp5763. Epub 2025 Jan 8.

Abstract

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.

MeSH terms

  • Animals
  • Fluorescence Resonance Energy Transfer* / methods
  • Humans
  • Mice
  • Opsins / genetics
  • Opsins / metabolism
  • Photons*

Substances

  • Opsins