A high-speed, bright, red fluorescent voltage sensor to detect neural activity

Connor Beck,Yiyang Gong

doi:10.1038/s41598-019-52370-8

Abstract

Genetically encoded voltage indicators (GEVIs) have emerged as a technology to optically record neural activity with genetic specificity and millisecond-scale temporal resolution using fluorescence microscopy. GEVIs have demonstrated ultra-fast kinetics and high spike detection fidelity in vivo, but existing red-fluorescent voltage indicators fall short of the response and brightness achieved by green fluorescent protein-based sensors. Furthermore, red-fluorescent GEVIs suffer from incomplete spectral separation from green sensors and blue-light-activated optogenetic actuators. We have developed Ace-mScarlet, a red fluorescent GEVI that fuses Ace2N, a voltage-sensitive inhibitory rhodopsin, with mScarlet, a bright red fluorescent protein (FP). Through fluorescence resonance energy transfer (FRET), our sensor detects changes in membrane voltage with high sensitivity and brightness and has kinetics comparable to the fastest green fluorescent sensors. Ace-mScarlet’s red-shifted absorption and emission spectra facilitate virtually complete spectral separation when used in combination with green-fluorescent sensors or with blue-light-sensitive sensors and rhodopsins. This spectral separation enables both simultaneous imaging in two separate wavelength channels and high-fidelity voltage recordings during simultaneous optogenetic perturbation.

Highlights

Encoded voltage indicators (GEVIs) have emerged as a technology to optically record neural activity with genetic specificity and millisecond-scale temporal resolution using fluorescence microscopy
Developed Genetically encoded voltage indicators (GEVIs) have millisecond timescale responses capable of producing high signal-to-noise ratio (SNR) readouts of single action potentials firing at frequencies as high as 100 Hz8–11
One broad family of GEVIs is the series of sensors that fused a green fluorescent protein (GFP) to a variety of voltage sensing domains (VSDs) to produce bright emission that relayed the action of the VSD

Summary

Introduction

Encoded voltage indicators (GEVIs) have emerged as a technology to optically record neural activity with genetic specificity and millisecond-scale temporal resolution using fluorescence microscopy. GEVIs have demonstrated ultra-fast kinetics and high spike detection fidelity in vivo, but existing red-fluorescent voltage indicators fall short of the response and brightness achieved by green fluorescent protein-based sensors. One broad family of GEVIs is the series of sensors that fused a green fluorescent protein (GFP) to a variety of voltage sensing domains (VSDs) to produce bright emission that relayed the action of the VSD These sensors have reported action potentials from neurons in culture, in slice, and in vivo. A high-throughput screening process developed VARNAM11, a fusion of the Ace-D81S rhodopsin voltage sensing domain and mRuby[3] These red-fluorescent GEVIs produced high SNR recordings at low to moderate excitation powers (15–100 mW/mm[2]). This photocurrent crosstalk arises from either direct excitation of the GEVI’s fluorophore by blue light (FlicR1) or weak activation of the rhodopsin by the yellow-green imaging illumination (VARNAM)

Methods

Results

Conclusion