Two-photon voltage imaging with rhodopsin-based sensors

Area of Science:

• Neuroscience
• Optical Imaging
• Molecular Biology

Background:

• Two-photon (2P) voltage imaging offers in-depth, high-resolution neuronal activity monitoring.
• Current 2P voltage imaging primarily uses ASAP-type sensors, while rhodopsin-based sensors are mainly used with one-photon (1P) illumination.

Purpose of the Study:

• To demonstrate the compatibility of rhodopsin-based genetically encoded voltage indicators (GEVIs) with 2P illumination.
• To engineer novel rhodopsin-based GEVIs for enhanced 2P voltage imaging.
• To assess the performance of these sensors in various biological preparations and conditions.

Main Methods:

• Rational engineering of a novel rhodopsin-based GEVI, named Jarvis.
• Testing Jarvis and existing FRET-opsin GEVIs (pAce, Voltron2) under 1P and 2P illumination.
• Comparing 2P scanless and fast 2P scanning illumination methods.
• Evaluating action potential detection in mouse hippocampal slices, zebrafish larvae, and awake mouse cortex.

Main Results:

• Demonstrated successful 2P illumination compatibility for rhodopsin-based GEVIs, including Jarvis, pAce, and Voltron2.
• Identified improved signal-to-noise ratio (SNR) for FRET-opsin GEVIs under low irradiance using scanless 2P illumination.
• Achieved high-SNR action potential detection at kilohertz rates using Jarvis and pAce in vitro and in vivo.

Conclusions:

• Rhodopsin-based GEVIs are effective tools for high-performance 2P voltage imaging.
• Scanless 2P illumination enhances SNR for FRET-opsin GEVIs.
• This work expands the toolkit for in-depth, high-speed neuronal activity monitoring in complex biological systems.