A general approach to engineer positive-going eFRET voltage indicators
Genetically encoded voltage indicators (GEVIs) enable cell-type-specific monitoring of electrical activity at subcellular resolution in neurons. Coupling bright fluorophores to rhodopsin-based GEVIs via electrochromic fluorescence resonance energy transfer (eFRET) enhances their brightness but results in 'negative-going' (brighter when hyperpolarized) signals. Further improvements of eFRET GEVIs are limited by the high background fluorescence inherent to negative-going sensors. Here we report a strategy for producing eFRET GEVIs with a positive-going fluorescence response, through rational manipulation of the native proton transport pathway in microbial rhodopsins. We used this strategy to transform the state-of-the-art eFRET GEVI Voltron into a positive-going sensor, Positron, with equivalent kinetics and sensitivity. We used Positron to image spiking and subthreshold voltage signals from several neurons simultaneously in zebrafish. We further applied our general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters. These sensors represent the first bright, positive-going GEVIs with sub-millisecond response kinetics.
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