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Two-photon voltage imaging with rhodopsin-based sensors
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Edité par CCSD -
International audience. Abstract The recent advances in sophisticated optical techniques, coupled with two-photon sensitive genetic voltage indicators (GEVIs), have enabled in-depth voltage imaging in vivo at single spike and single-cell resolution. To date, these results have been only achieved using ASAP-type sensors, as the complex photocycle of rhodopsin-based voltage indicators posed challenges for their two-photon use, restricting their application to one-photon approaches. In this work, we demonstrate that rhodopsin-based GEVIs (FRET-opsin) can be used under two-photon illumination when their peculiar light intensity dependence of kinetics and sensitivity are considered. We rationally engineer a fully genetically-encoded, rhodopsin-based voltage indicator with the brightest known fluorophore AaFP1, Jarvis, and demonstrate its utility under both one- and two-photon illumination. We also showed two-photon usability of the similar FRET-opsin sensor pAce. Our comparison of 2P scanless with fast 2P scanning illumination revealed that the latter approach is less suitable for this class of indicators and, on the contrary, both sensors responded well when scanless approaches were used. Furthermore, utilising Jarvis, we demonstrated high-fidelity, high-SNR action potential detection at kilohertz-imaging rates both in mouse hippocampal slices and in zebrafish larvae. To the best of our knowledge, this study represents the first report of a fully genetically-encoded rhodopsin-based voltage indicator for high contrast action potential detection under two-photon illumination in vitro and in vivo .
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