Photophysics-informed Two-photon Voltage Imaging Using FRET-opsin Voltage Indicators

Overview

Journal Sci Adv

Specialties Biology
Science

Date 2025 Jan 8

PMID 39772682

Authors

F Phil Brooks 3rd

Daozheng Gong

Hunter C Davis

Pojeong Park

Yitong Qi

Adam E Cohen

Affiliations

Soon will be listed here.

Abstract

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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References

Landau A, Park P, Wong-Campos J, Tian H, Cohen A, Sabatini B . Dendritic branch structure compartmentalizes voltage-dependent calcium influx in cortical layer 2/3 pyramidal cells. Elife. 2022; 11. PMC: 8979587. DOI: 10.7554/eLife.76993. View

Maclaurin D, Venkatachalam V, Lee H, Cohen A . Mechanism of voltage-sensitive fluorescence in a microbial rhodopsin. Proc Natl Acad Sci U S A. 2013; 110(15):5939-44. PMC: 3625274. DOI: 10.1073/pnas.1215595110. View

Zou P, Zhao Y, Douglass A, Hochbaum D, Brinks D, Werley C . Bright and fast multicoloured voltage reporters via electrochromic FRET. Nat Commun. 2014; 5:4625. PMC: 4134104. DOI: 10.1038/ncomms5625. View

Abdelfattah A, Valenti R, Zheng J, Wong A, Podgorski K, Koyama M . A general approach to engineer positive-going eFRET voltage indicators. Nat Commun. 2020; 11(1):3444. PMC: 7351947. DOI: 10.1038/s41467-020-17322-1. View

Kralj J, Douglass A, Hochbaum D, Maclaurin D, Cohen A . Optical recording of action potentials in mammalian neurons using a microbial rhodopsin. Nat Methods. 2011; 9(1):90-5. PMC: 3248630. DOI: 10.1038/nmeth.1782. View

Hou J, Venkatachalam V, Cohen A . Temporal dynamics of microbial rhodopsin fluorescence reports absolute membrane voltage. Biophys J. 2014; 106(3):639-48. PMC: 3945107. DOI: 10.1016/j.bpj.2013.11.4493. View

Adam Y . All-optical electrophysiology in behaving animals. J Neurosci Methods. 2021; 353:109101. DOI: 10.1016/j.jneumeth.2021.109101. View

Gong Y, Wagner M, Li J, Schnitzer M . Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors. Nat Commun. 2014; 5:3674. PMC: 4247277. DOI: 10.1038/ncomms4674. View

Penzkofer A, Silapetere A, Hegemann P . Photocycle dynamics of the Archaerhodopsin 3 based fluorescent voltage sensor Archon2. J Photochem Photobiol B. 2021; 225:112331. DOI: 10.1016/j.jphotobiol.2021.112331. View

10.

Hendler R, Shrager R, Bose S . Theory and procedures for finding a correct kinetic model for the bacteriorhodopsin photocycle. J Phys Chem B. 2013; 105(16):3319-28. DOI: 10.1021/jp002362z. View

11.

Azimi Hashemi N, Bergs A, Schuler C, Scheiwe A, Costa W, Bach M . Rhodopsin-based voltage imaging tools for use in muscles and neurons of . Proc Natl Acad Sci U S A. 2019; 116(34):17051-17060. PMC: 6708366. DOI: 10.1073/pnas.1902443116. View

12.

Abdelfattah A, Kawashima T, Singh A, Novak O, Liu H, Shuai Y . Bright and photostable chemigenetic indicators for extended in vivo voltage imaging. Science. 2019; 365(6454):699-704. DOI: 10.1126/science.aav6416. View

13.

Goldey G, Roumis D, Glickfeld L, Kerlin A, Reid R, Bonin V . Removable cranial windows for long-term imaging in awake mice. Nat Protoc. 2014; 9(11):2515-2538. PMC: 4442707. DOI: 10.1038/nprot.2014.165. View

14.

Kubel J, Chenchiliyan M, Ooi S, Gustavsson E, Isaksson L, Kuznetsova V . Transient IR spectroscopy identifies key interactions and unravels new intermediates in the photocycle of a bacterial phytochrome. Phys Chem Chem Phys. 2020; 22(17):9195-9203. DOI: 10.1039/c9cp06995j. View

15.

Liu S, Lin C, Xu Y, Luo H, Peng L, Zeng X . A far-red hybrid voltage indicator enabled by bioorthogonal engineering of rhodopsin on live neurons. Nat Chem. 2021; 13(5):472-479. DOI: 10.1038/s41557-021-00641-1. View

16.

Bayraktar H, Fields A, Kralj J, Spudich J, Rothschild K, Cohen A . Ultrasensitive measurements of microbial rhodopsin photocycles using photochromic FRET. Photochem Photobiol. 2011; 88(1):90-7. PMC: 3253248. DOI: 10.1111/j.1751-1097.2011.01011.x. View

17.

Chien M, Brinks D, Testa-Silva G, Tian H, Phil Brooks 3rd F, Adam Y . Photoactivated voltage imaging in tissue with an archaerhodopsin-derived reporter. Sci Adv. 2021; 7(19). PMC: 8099184. DOI: 10.1126/sciadv.abe3216. View

18.

Charan K, Li B, Wang M, Lin C, Xu C . Fiber-based tunable repetition rate source for deep tissue two-photon fluorescence microscopy. Biomed Opt Express. 2018; 9(5):2304-2311. PMC: 5946790. DOI: 10.1364/BOE.9.002304. View

19.

Gong Y, Huang C, Li J, Grewe B, Zhang Y, Eismann S . High-speed recording of neural spikes in awake mice and flies with a fluorescent voltage sensor. Science. 2015; 350(6266):1361-6. PMC: 4904846. DOI: 10.1126/science.aab0810. View

20.

Huang C, Luo J, Woo S, Roitman L, Li J, Pieribone V . Dopamine-mediated interactions between short- and long-term memory dynamics. Nature. 2024; 634(8036):1141-1149. PMC: 11525173. DOI: 10.1038/s41586-024-07819-w. View