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Abstract Opsin‐based transmembrane voltage sensors (OTVSs) are increasingly important tools for neuroscience enabling neural function in complex brain circuits to be explored in live, behaving animals. However, the visible wavelengths required for fluorescence excitation of the current generation of OTVSs limit optogenetic imaging in the brain to depths of only a few mm due to the strong absorption and scattering of visible light by biological tissues. We report that substitution of the native A1 retinal chromophore of the widely used QuasAr1/2 OTVSs with the retinal analog MMAR containing a methylamino‐modified dimethylphenyl ring results in over a 100‐nm redshift of the maxima of the absorption and fluorescence emission bands to near 700 and 840 nm, respectively. FT‐Raman spectroscopy reveals that at pH 7 QuasAr1 with both the A1 and MMAR chromophores possess predominantly an all‐transprotonated Schiff base configuration with the MMAR chromophore exhibiting increased torsion of the polyene single‐/double‐bond system similar to the O‐intermediate of the BR photocycle. In contrast, the A1 and the MMAR chromophores of QuasAr2 exist partially in a 13‐cisPSB configuration. These results demonstrate that QuasArs containing the MMAR chromophore are attractive candidates for use as NIR‐OTVSs, especially for applications such as deep brain imaging.
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This content will become publicly available on January 10, 2026
Photophysics-informed two-photon voltage imaging using FRET-opsin voltage indicators
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 quenching 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 Voltron2. We found that the previously reported negative-going voltage sensitivities of both GEVIs came from photocycle intermediates, not from the opsin ground states. The voltage sensitivities of both GEVIs were nonlinear functions of illumination intensity; for Voltron1, the sensitivity reversed the sign under low-intensity illumination. Using photocycle-optimized 2P illumination protocols, we demonstrate 2P voltage imaging with Voltron2 in the barrel cortex of a live mouse. These results open the door to high-speed 2P voltage imaging of FRET-opsin GEVIs in vivo.
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- Award ID(s):
- 2121044
- PAR ID:
- 10591577
- Publisher / Repository:
- PubMedCentral
- Date Published:
- Journal Name:
- Science Advances
- Volume:
- 11
- Issue:
- 2
- ISSN:
- 2375-2548
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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