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Optical constraints on two-photon voltage imaging.

ABSTRACT: Genetically encoded voltage indicators (GEVIs) are a valuable tool for studying neural circuits in vivo, but the relative merits of one-photon (1P) vs. two-photon (2P) voltage imaging are not well characterized. Here we compare the photophysical and imaging properties of commonly used GEVIs under 1P and 2P excitation. 2P excitation requires ~10⁴-fold more illumination power per cell to produce comparable photon count rates to 1P excitation, driving a stringent tradeoff between shot noise and tissue photodamage.

SUBMITTER: Davis HC

PROVIDER: S-EPMC10680948 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Optical constraints on two-photon voltage imaging.

Brooks F Phil FP Davis Hunter C HC Wong-Campos J David JD Cohen Adam E AE

bioRxiv : the preprint server for biology 20240517

<h4>Significance</h4>Genetically encoded voltage indicators (GEVIs) are a valuable tool for studying neural circuits <i>in vivo</i>, but the relative merits and limitations of one-photon (1P) vs. two-photon (2P) voltage imaging are not well characterized.<h4>Aim</h4>We consider the optical and biophysical constraints particular to 1P and 2P voltage imaging and compare the imaging properties of commonly used GEVIs under 1P and 2P excitation.<h4>Approach</h4>We measure brightness and voltage sensi ...[more]

PMID: 38014011

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Project description:Optical methods that rely on fluorescence for mapping changes in neuronal membrane potential in the brains of awake animals provide a powerful way to interrogate the activity of neurons that underlie neural computations ranging from sensation and perception to learning and memory. To achieve this goal, fluorescent indicators should be bright, highly sensitive to small changes in membrane potential, nontoxic, and excitable with infrared light. We report a new class of fluorescent, voltage-sensitive dyes: sulfonated rhodamine voltage reporters (sRhoVR), synthetic fluorophores with high voltage sensitivity, excellent two-photon performance, and compatibility in intact mouse brains. sRhoVR dyes are based on a tetramethyl rhodamine fluorophore coupled to a phenylenevinylene molecular wire/diethyl aniline voltage-sensitive domain. When applied to cells, sRhoVR dyes localize to the plasma membrane and respond to membrane depolarization with a fluorescence increase. The best of the new dyes, sRhoVR 1, displays a 44% ?F/F increase in fluorescence per 100 mV change, emits at 570 nm, and possesses excellent two-photon absorption of approximately 200 GM at 840 nm. sRhoVR 1 can detect action potentials in cultured rat hippocampal neurons under both single- and two-photon illumination with sufficient speed and sensitivity to report on action potentials in single trials, without perturbing underlying physiology or membrane properties. The combination of speed, sensitivity, and brightness under two-photon illumination makes sRhoVR 1 a promising candidate for in vivo imaging in intact brains. We show sRhoVR powerfully complements electrode-based modes of neuronal activity recording in the mouse brain by recording neuronal transmembrane potentials from the neuropil of layer 2/3 of the mouse barrel cortex in concert with extracellularly recorded local field potentials (LFPs). sRhoVR imaging reveals robust depolarization in response to whisker stimulation; concurrent electrode recordings reveal negative deflections in the LFP recording, consistent with the canonical thalamocortical response. Importantly, sRhoVR 1 can be applied in mice with chronic optical windows, presaging its utility in dissecting and resolving voltage dynamics using two-photon functional imaging in awake, behaving animals.

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Optical constraints on two-photon voltage imaging.

Publications

Optical constraints on two-photon voltage imaging.

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