Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits its speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and computationally recovers high-resolution images, attaining voxel rates of over 1 billion Hz in structured samples. Using a static image as a prior for recording neural activity, we imaged visually-evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 μm and frame-rates over 1 kHz. Dendritic glutamate transients in anaesthetized mice are synchronized within spatially-contiguous domains spanning tens of microns at frequencies ranging from 1-100 Hz. We demonstrate millisecond-resolved recordings of acetylcholine and voltage indicators, 3D single-particle tracking, and imaging in Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

We present the design, synthesis, and application of a new family of fluorescent voltage indicators based on isomerically pure tetramethylrhodamines. These new Rhodamine Voltage Reporters, or RhoVRs, use photoinduced electron transfer (PeT) as a trigger for voltage sensing, display excitation and emission profiles in the green to orange region of the visible spectrum, demonstrate high sensitivity to membrane potential changes (up to 47% ΔF/F per 100 mV), and employ a tertiary amide derived from sarcosine, which aids in membrane localization and simultaneously simplifies the synthetic route to the voltage sensors. The most sensitive of the RhoVR dyes, RhoVR 1, features a methoxy-substituted diethylaniline donor and phenylenevinylene molecular wire in the 5′-position of the rhodamine aryl ring, the highest voltage-sensitivity to date for red-shifted PeT-based voltage sensors, and is compatible with simultaneous imaging alongside GFP-based indicators. The discoveries that sarcosine-based tertiary amides in the context of molecular wire voltage indicators prevent dye internalization, along with the improved voltage sensitivity of 5′-substituted voltage indicators should be broadly applicable to other types of PeT-based voltage-sensitive fluorophores.

Voltage-sensitive fluorophores enable the direct visualization of membrane potential changes in living systems. To pair the speed and sensitivity of chemical synthesized fluorescent indicators with cell-type specific genetic methods, we here develop Rhodamine-based Voltage Reporters (RhoVR) that can be covalently tethered to genetically-encoded, self-labeling enzymes. These chemical-genetic hybrids feature a photoinduced electron transfer (PeT) triggered RhoVR voltagesensitive indicator coupled to a chloroalkane HaloTag ligand through a long, water-soluble polyethyleneglycol (PEG) linker (RhoVR-Halos). When applied to cells, RhoVR-Halos selectively and covalently bind to surface-expressed HaloTag enzyme on genetically modified cells. RhoVR-Halos maintain high voltage sensitivities-up to 34% ΔF/F per 100 mV-and fast response times typical of untargeted RhoVRs, while gaining the selectivity typical of genetically encodable voltage indicators. We show that RhoVR-Halos can record action potentials in single trials from cultured rat hippocampal neurons and can be used in concert with green-fluorescent Ca 2+ indicators like GCaMP to provide simultaneous voltage and Ca 2+ imaging. In brain slice, RhoVR-Halos provide exquisite labeling of defined cells and can be imaged using epifluorescence, confocal, or two-photon microscopy. Using high-speed epifluorescence microscopy, RhoVR-Halos provide a read out of action potentials from labeled cortical neurons in rat brain slice, without the need for trial averaging. These results demonstrate the potential of hybrid chemical-genetic voltage indicators to combine the optical performance of small-molecule chromophores with the inherent selectivity of genetically-encodable systems, permitting imaging modalities inaccessible to either technique individually.

Voltage-sensitive fluorophores enable the direct visualization of membrane potential changes in living systems. To pair the speed and sensitivity of chemical synthesized fluorescent indicators with cell-type specific genetic methods, we here develop Rhodamine-based Voltage Reporters (RhoVR) that can be covalently tethered to genetically-encoded, self-labeling enzymes. These chemical-genetic hybrids feature a photoinduced electron transfer (PeT) triggered RhoVR voltage-sensitive indicator coupled to a chloroalkane HaloTag ligand through a long, water-soluble polyethyleneglycol (PEG) linker (RhoVR-Halos). When applied to cells, RhoVR-Halos selectively and covalently bind to surface-expressed HaloTag enzyme on genetically modified cells. RhoVR-Halos maintain high voltage sensitivities—up to 34% ΔF/F per 100 mV—and fast response times typical of untargeted RhoVRs, while gaining the selectivity typical of genetically encodable voltage indicators. We show that RhoVR-Halos can record action potentials in single trials from cultured rat hippocampal neurons and can be used in concert with green-fluorescent Ca²⁺ indicators like GCaMP to provide simultaneous voltage and Ca²⁺ imaging. In brain slice, RhoVR-Halos provide exquisite labeling of defined cells and can be imaged using epifluorescence, confocal, or two-photon microscopy. Using high-speed epifluorescence microscopy, RhoVR-Halos provide a read out of action potentials from labeled cortical neurons in rat brain slice, without the need for trial averaging. These results demonstrate the potential of hybrid chemical-genetic voltage indicators to combine the optical performance of small-molecule chromophores with the inherent selectivity of genetically-encodable systems, permitting imaging modalities inaccessible to either technique individually.

In the version of this Article originally published, the authors incorrectly gave the name of a funding source as the 'Klingenstein-Simons Foundation'; the correct name is the 'Klingenstein-Simons Fellowship Award in Neurosciences'. This has now been amended in all online versions of the Article.