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

Autor:	Brooks FP 3rd; Department of Chemistry and Chemical Biology, Harvard University., Davis HC; Department of Chemistry and Chemical Biology, Harvard University., Park P; Department of Chemistry and Chemical Biology, Harvard University., Qi Y; Department of Chemistry and Chemical Biology, Harvard University., Cohen AE; Department of Chemistry and Chemical Biology, Harvard University.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2024 Apr 02. Date of Electronic Publication: 2024 Apr 02.
DOI:	10.1101/2024.04.01.587540
Abstrakt:	Microbial rhodopsin-derived genetically encoded voltage indicators (GEVIs) are powerful tools for mapping bioelectrical dynamics in cell culture and in live animals. Förster resonance energy transfer (FRET)-opsin GEVIs use voltage-dependent changes in opsin absorption to modulate the fluorescence of an attached fluorophore, achieving high brightness, speed, and voltage sensitivity. However, the voltage sensitivity of most FRET-opsin GEVIs has been reported to decrease or vanish under two-photon (2P) excitation. Here we investigated the photophysics of the FRET-opsin GEVIs Voltron1 and 2. We found that the voltage sensitivity came from a photocycle intermediate, not from the opsin ground state. The voltage sensitivities of both GEVIs were nonlinear functions of illumination intensity; for Voltron1, the sensitivity reversed sign under low-intensity illumination. Using photocycle-optimized 2P illumination protocols, we demonstrate 2P voltage imaging with Voltron2 in barrel cortex of a live mouse. These results open the door to high-speed 2P voltage imaging of FRET-opsin GEVIs in vivo . Competing Interests: The authors declare that they have no competing interests.
Databáze:	MEDLINE
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