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

Area of Science:

• Neuroscience
• Biophysics
• Molecular Biology

Background:

• Microbial rhodopsin-derived genetically encoded voltage indicators (GEVIs) are crucial for mapping bioelectrical activity.
• Förster resonance energy transfer (FRET)-opsin GEVIs offer high brightness, speed, and voltage sensitivity.
• A significant limitation is the reduced voltage sensitivity of FRET-opsin GEVIs under two-photon (2P) excitation.

Purpose of the Study:

• To investigate the photophysics of FRET-opsin GEVIs Voltron1 and Voltron2.
• To understand the reasons behind decreased voltage sensitivity under 2P excitation.
• To develop optimized protocols for effective 2P voltage imaging in vivo.

Main Methods:

• Photophysical characterization of Voltron1 and Voltron2.
• Analysis of voltage sensitivity as a function of illumination intensity.
• Development and application of photocycle-optimized 2P illumination protocols.
• In vivo 2P voltage imaging in a live mouse model.

Main Results:

• The observed voltage sensitivity in FRET-opsin GEVIs originates from photocycle intermediates, not ground states.
• Voltage sensitivity is a nonlinear function of illumination intensity, with sign reversal possible under low intensity.
• Photocycle-optimized 2P illumination protocols were successfully developed.
• High-speed 2P voltage imaging was demonstrated in vivo using Voltron2 in mouse brain.

Conclusions:

• The photophysics of FRET-opsin GEVIs under 2P excitation are complex and influenced by photocycle intermediates and illumination intensity.
• Optimized 2P illumination protocols can overcome previous limitations, enabling effective in vivo voltage imaging.
• These findings pave the way for advanced high-speed 2P voltage imaging applications in neuroscience.