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

Area of Science:

• Neuroscience
• Biophysics
• Molecular Imaging

Background:

• Genetically encoded voltage indicators (GEVIs) derived from microbial rhodopsins are crucial for mapping bioelectrical activity.
• Förster resonance energy transfer (FRET)-opsin GEVIs offer high brightness, speed, and voltage sensitivity but often lose sensitivity under two-photon (2P) excitation.

Approach:

• Investigated the photophysics of FRET-opsin GEVIs Voltron1 and Voltron2.
• Identified a photocycle intermediate, not the opsin ground state, as the source of voltage sensitivity.
• Analyzed the nonlinear relationship between illumination intensity and voltage sensitivity, noting sign reversal in Voltron1 at low intensities.

Key Points:

• Voltage sensitivity in Voltron1 and Voltron2 originates from a photocycle intermediate.
• GEVI sensitivity is a nonlinear function of illumination intensity.
• Photocycle-optimized 2P illumination protocols were developed.

Conclusions:

• Demonstrated successful 2P voltage imaging of Voltron2 in the mouse barrel cortex.
• These findings pave the way for high-speed 2P voltage imaging of FRET-opsin GEVIs in vivo.
• Understanding the photophysics is key to overcoming limitations of GEVIs under 2P microscopy.