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The membrane potential is a critical aspect of cellular physiology, essential for maintaining homeostasis, facilitating signal transduction, and driving various cellular processes. While the resting membrane potential (RMP) represents a key physiological parameter, membrane potential fluctuations, such as depolarization and hyperpolarization, are equally vital in understanding dynamic cellular behavior. Traditional techniques, such as microelectrodes and patch-clamp methods, offer valuable insights but are invasive and less suited for high-throughput applications. Recent advances in voltage indicators, including fast and slow dyes, and novel imaging modalities such as second harmonic generation (SHG) and photoacoustic imaging, enable noninvasive, high-resolution measurement of both RMP and membrane potential dynamics. This review explores the mechanisms, development, and applications of these tools, emphasizing their transformative potential in neuroscience and cellular electrophysiology research.

Voltage stability improvement is a challenging issue in planning and security assessment of power systems. As modern systems are being operated under heavily stressed conditions with reduced stability margins, incorporation of voltage stability criteria in the operation of power systems began receiving great attention. This study presents a novel voltage stability constrained optimal power flow (VSC-OPF) approach based on static line voltage stability indices to simultaneously improve voltage stability and minimise power system losses under stressed and contingency conditions. The proposed methodology uses a voltage collapse proximity indicator (VCPI) to provide important information about the proximity of the system to voltage instability. The VCPI index is incorporated into the optimal power flow (OPF) formulation in two ways; first it can be added as a new voltage stability constraint in the OPF constraints, or used as a voltage stability objective function. The proposed approach has been evaluated on the standard IEEE 30-bus and 57-bus test systems under different cases and compared with two well proved VSC-OPF approaches based on the bus voltage indicator L-index and the minimum singular value. The simulation results are promising and demonstrate the effectiveness of the proposed VSC-OPF based on the line voltage stability index.

Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

A technology that simultaneously records membrane potential from multiple neurons in behaving animals will have a transformative effect on neuroscience research 1 , 2 . Genetically encoded voltage indicators are a promising tool for these purposes; however, these have so far been limited to single-cell recordings with a marginal signal-to-noise ratio in vivo 3 – 5 . Here we developed improved near-infrared voltage indicators, high-speed microscopes and targeted gene expression schemes that enabled simultaneous in vivo recordings of supra- and subthreshold voltage dynamics in multiple neurons in the hippocampus of behaving mice. The reporters revealed subcellular details of back-propagating action potentials and correlations in subthreshold voltage between multiple cells. In combination with stimulation using optogenetics, the reporters revealed changes in neuronal excitability that were dependent on the behavioural state, reflecting the interplay of excitatory and inhibitory synaptic inputs. These tools open the possibility for detailed explorations of network dynamics in the context of behaviour. Fig. 1 Photoactivated QuasAr3 (paQuasAr3) reports neuronal activity in vivo. a , Schematic of the paQuasAr3 construct. b , Photoactivation by blue light enhanced voltage signals excited by red light in cultured neurons that expressed paQuasAr3 (representative example of n  = 4 cells). c , Model of the photocycle of paQuasAr3. d , Confocal images of sparsely expressed paQuasAr3 in brain slices. Scale bars, 50 μm. Representative images, experiments were repeated in n  = 3 mice. e , Simultaneous fluorescence and patch-clamp recordings from a neuron expressing paQuasAr3 in acute brain slice. Top, magnification of boxed regions. Schematic shows brain slice, patch pipette and microscope objective. f , Simultaneous fluorescence and patch-clamp recordings of inhibitory post synaptic potentials in an L2–3 neuron induced by electrical stimulation of L5–6 in acute slice. g , Normalized change in fluorescence (Δ F / F ) and SNR of optically recorded post-synaptic potentials (PSPs) as a function of the amplitude of the post-synaptic potentials. The voltage sensitivity was Δ F / F  = 40 ± 1.7% per 100 mV. The SNR was 0.93 ± 0.07 per 1 mV in a 1-kHz bandwidth ( n  = 42 post-synaptic potentials from 5 cells, data are mean ± s.d.). Schematic shows brain slice, patch pipette, field stimulation electrodes and microscope objective. h , Optical measurements of paQuasAr3 fluorescence in the CA1 region of the hippocampus (top) and glomerular layer of the olfactory bulb (bottom) of anaesthetized mice (representative traces from n  = 7 CA1 cells and n  = 13 olfactory bulb cells, n  = 3 mice). Schematics show microscope objective and the imaged brain region. i , STA fluorescence from 88 spikes in a CA1 oriens neuron. j , Frames from the STA video showing the delay in the back-propagating action potential in the dendrites relative to the soma. k , Sub-Nyquist fitting of the action potential delay and width shows electrical compartmentalization in the dendrites. Experiments in k – m were repeated in n  = 2 cells from n  = 2 mice. A combination of improved near-infrared voltage indicators, high-speed microscopes and targeted gene expression schemes enabled simultaneous in vivo optogenetic control and recording of voltage dynamics in multiple neurons in the hippocampus of behaving mice.

We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.

Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence–voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings. The ASAP4 family of genetically encoded voltage indicators allows recording of action potentials and subthreshold activity with either one- or two-photon microscopy over extended periods of time.

Monitoring spiking activity across large neuronal populations at behaviorally relevant timescales is critical for understanding neural circuit function. Unlike calcium imaging, voltage imaging requires kilohertz sampling rates that reduce fluorescence detection to near shot-noise levels. High-photon flux excitation can overcome photon-limited shot noise, but photobleaching and photodamage restrict the number and duration of simultaneously imaged neurons. We investigated an alternative approach aimed at low two-photon flux, which is voltage imaging below the shot-noise limit. This framework involved developing positive-going voltage indicators with improved spike detection (SpikeyGi and SpikeyGi2); a two-photon microscope (‘SMURF’) for kilohertz frame rate imaging across a 0.4 mm × 0.4 mm field of view; and a self-supervised denoising algorithm (DeepVID) for inferring fluorescence from shot-noise-limited signals. Through these combined advances, we achieved simultaneous high-speed deep-tissue imaging of more than 100 densely labeled neurons over 1 hour in awake behaving mice. This demonstrates a scalable approach for voltage imaging across increasing neuronal populations. A suite of tools including positive-going voltage indicators, a high-speed two-photon microscope, and denoising software enables prolonged imaging of electrical activity in neurons with limited toxicity.

A longstanding goal in neuroscience has been to image membrane voltage across a population of individual neurons in an awake, behaving mammal. Here we describe a genetically encoded fluorescent voltage indicator, SomArchon, which exhibits millisecond response times and is compatible with optogenetic control, and which increases the sensitivity, signal-to-noise ratio, and number of neurons observable several-fold over previously published fully genetically encoded reagents 1 – 8 . Under conventional one-photon microscopy, SomArchon enables the routine population analysis of around 13 neurons at once, in multiple brain regions (cortex, hippocampus, and striatum) of head-fixed, awake, behaving mice. Using SomArchon, we detected both positive and negative responses of striatal neurons during movement, as previously reported by electrophysiology but not easily detected using modern calcium imaging techniques 9 – 11 , highlighting the power of voltage imaging to reveal bidirectional modulation. We also examined how spikes relate to the subthreshold theta oscillations of individual hippocampal neurons, with SomArchon showing that the spikes of individual neurons are more phase-locked to their own subthreshold theta oscillations than to local field potential theta oscillations. Thus, SomArchon reports both spikes and subthreshold voltage dynamics in awake, behaving mice. A genetically encoded fluorescent voltage indicator, SomArchon, is used to image changes in membrane voltage from many neurons simultaneously in multiple brain regions of awake, behaving mice.

Video-based screening of pooled libraries is a powerful approach for directed evolution of biosensors because it enables selection along multiple dimensions simultaneously from large libraries. Here we develop a screening platform, Photopick, which achieves precise phenotype-activated photoselection over a large field of view (2.3 × 2.3 mm, containing >10 3  cells, per shot). We used the Photopick platform to evolve archaerhodopsin-derived genetically encoded voltage indicators (GEVIs) with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). These GEVIs gave improved signals in cultured neurons and in live mouse brains. By combining targeted in vivo optogenetic stimulation with high-precision voltage imaging, we characterized inhibitory synaptic coupling between individual cortical NDNF (neuron-derived neurotrophic factor) interneurons, and excitatory electrical synapses between individual hippocampal parvalbumin neurons. The QuasAr6 GEVIs are powerful tools for all-optical electrophysiology and the Photopick approach could be adapted to evolve a broad range of biosensors. The Photopick platform, which can be used for phenotype-activated cell selection, was used to develop the improved voltage sensors QuasAr6a and QuasAr6b. These GEVIs offer improved signals and are useful for all-optical electrophysiology.

Membrane potential is a key aspect of cellular signalling and is dynamically regulated by an array of ion-selective pumps and channels. Fluorescent voltage indicators enable non-invasive optical recording of the cellular membrane potential with high spatial resolution. Here, we report a palette of bright and sensitive hybrid voltage indicators (HVIs) with fluorescence intensities sensitive to changes in membrane potential via electrochromic Förster resonance energy transfer. Enzyme-mediated site-specific incorporation of a probe, followed by an inverse-electron-demand Diels–Alder cycloaddition, was used to create enhanced voltage-sensing rhodopsins with hybrid dye–protein architectures. The most sensitive indicator, HVI-Cy3, displays high voltage sensitivity (−39% ΔF/F0 per 100 mV) and millisecond response kinetics, enabling optical recording of action potentials at a sampling rate of 400 Hz over 10 min across a large neuronal population. The far-red indicator HVI-Cy5 could be paired with optogenetic actuators and green/red-emitting fluorescent indicators, allowing an all-optical investigation of neuronal electrophysiology.Voltage imaging is a powerful technique for studying electrical signalling in neurons. A palette of bright and sensitive voltage indicators has now been developed via enzyme-mediated ligation and Diels–Alder cycloaddition. Among these, a far-red indicator faithfully reports neuronal action potential dynamics with an excitation spectrum orthogonal to optogenetic actuators and green/red-emitting biosensors.