Voltage Imaging Research Articles

Orange/far-red hybrid voltage indicators with reduced phototoxicity enable reliable long-term imaging in neurons and cardiomyocytes

Hybrid voltage indicators (HVIs) are chemogenetic sensors that combines the superior photophysical properties of organic dyes and the genetic targetability of protein sensors to report transient membrane voltage changes. They exhibit boosted sensitivity in excitable cells such as neurons and cardiomyocytes. However, the voltage signals recorded during long-term imaging are severely diminished or distorted due to phototoxicity and photobleaching issues. To capture stable electrophysiological activities over a long time, we employ cyanine dyes conjugated with a cyclooctatetraene (COT) molecule as the fluorescence reporter of HVI. The resulting orange-emitting HVI-COT-Cy3 enables high-fidelity voltage imaging for up to 30 min in cultured primary neurons with a sensitivity of ~ -30% ΔF/F0 per action potential (AP). It also maximally preserves the signal of individual APs in cardiomyocytes. The far-red-emitting HVI-COT-Cy5 allows two-color voltage/calcium imaging with GCaMP6s in neurons and cardiomyocytes for 15 min. We leverage the HVI-COT series with reduced phototoxicity and photobleaching to evaluate the impact of drug candidates on the electrophysiology of excitable cells.

Near-infrared voltage-sensitive dyes based on chromene donor

Voltage-sensitive dyes (VSDs) are used to image electrical activity in cells and tissues with submillisecond time resolution. Most of these fast sensors are constructed from push-pull chromophores whose fluorescence spectra are modulated by the electric field across the cell membrane. It was found that the substitution of naphthalene with chromene produces a 60 to 80 nm red-shift in absorption and emission spectra while maintaining fluorescence quantum efficiency and voltage sensitivity. One dye was applied to ex vivo murine heart with excitation at 730 nm, by far the longest wavelength reported in voltage imaging. This VSD resolves cardiac action potentials in single trials with 12% ΔF/F per action potential. The well-separated excitation spectra between these long-wavelength VSDs and channelrhodopsin (ChR2) enabled monitoring of action potential propagation in ChR2 hearts without any perturbation of electrical dynamics. Importantly, by employing spatially localized optogenetic manipulation, action potential dynamics can be assessed in an all-optical fashion with no artifact related to optical cross-talk between the reporter and actuator. These new environmentally sensitive chromene-based chromophores are also likely to have applications outside voltage imaging.

Theta and gamma rhythmic coding through two spike output modes in the hippocampus during spatial navigation

Hippocampal CA1 neurons generate single spikes and stereotyped bursts of spikes. However, it is unclear how individual neurons dynamically switch between these output modes and whether these two spiking outputs relay distinct information. We performed extracellular recordings in spatially navigating rats and cellular voltage imaging and optogenetics in awake mice. We found that spike bursts are preferentially linked to cellular and network theta rhythms (3-12Hz) and encode an animal's position via theta phase precession, particularly as animals are entering a place field. In contrast, single spikes exhibit additional coupling to gamma rhythms (30-100Hz), particularly as animals leave a place field. Biophysical modeling suggests that intracellular properties alone are sufficient to explain the observed input frequency-dependent spike coding. Thus, hippocampal neurons regulate the generation of bursts and single spikes according to frequency-specific network and intracellular dynamics, suggesting that these spiking modes perform distinct computations to support spatial behavior.

Supertemporal Resolution Imaging of Membrane Potential via Stroboscopic Microscopy.

Membrane potential and its fluctuation are fundamental biophysical phenomena essential to cellular activities and functions. Compared to traditional electrode-based techniques, the optical recording via developed genetically encoded voltage indicators (GEVIs) offers a combination of noninvasiveness, high spatial resolution, and increased measurement throughput. However, its application is limited by the insufficient acquisition rate and time accuracy of the camera. Here we design and apply a stroboscopic illumination scheme to boost the temporal resolution of voltage imaging, while simultaneously eliminating the artifacts caused by nonsynchronized exposure in the rolling-shutter mode. We demonstrate that commonly used GEVIs are compatible with stroboscopic voltage imaging (SVI), and our SVI scheme offers a 5-fold faster acquisition frame rate than that of conventional continuous illumination. The GEVIs tested maintain high sensitivities in the SVI mode, supporting faithful reports of intracellular depolarization waveform and intercellular gap junction-mediated depolarization coupling in human embryonic kidney 293T (HEK 293T) cell populations. SVI allows resolving the action potential (AP) waveform with less distortion and mapping action potential initiation and propagation dynamics in cultured neurons in kilohertz, beyond the restriction from the camera in the field of view.

Voltage-Seq: all-optical postsynaptic connectome-guided single-cell transcriptomics

Understanding the routing of neuronal information requires the functional characterization of connections. Neuronal projections recruit large postsynaptic ensembles with distinct postsynaptic response types (PRTs). PRT is typically probed by low-throughput whole-cell electrophysiology and is not a selection criterion for single-cell RNA-sequencing (scRNA-seq). To overcome these limitations and target neurons based on specific PRTs for soma harvesting and subsequent scRNA-seq, we created Voltage-Seq. We established all-optical voltage imaging and recorded the PRT of 8,347 neurons in the mouse periaqueductal gray (PAG) evoked by the optogenetic activation of ventromedial hypothalamic (VMH) terminals. PRTs were classified and spatially resolved in the entire VMH-PAG connectome. We built an onsite analysis tool named VoltView to navigate soma harvesting towards target PRTs guided by a classifier that used the VMH-PAG connectome database as a reference. We demonstrated Voltage-seq by locating VMH-driven γ-aminobutyric acid-ergic neurons in the PAG, guided solely by the onsite classification in VoltView.

Velocity of conduction between columns and layers in barrel cortex reported by parvalbumin interneurons.

Inhibitory interneurons expressing parvalbumin (PV) play critical roles throughout the brain. Their rapid spiking enables them to control circuit dynamics on a millisecond time scale, and the timing of their activation by different excitatory pathways is critical to these functions. We used a genetically encoded hybrid voltage sensor to image PV interneuron voltage changes with sub-millisecond precision in primary somatosensory barrel cortex (BC) of adult mice. Electrical stimulation evoked depolarizations with a latency that increased with distance from the stimulating electrode, allowing us to determine conduction velocity. Spread of responses between cortical layers yielded an interlaminar conduction velocity and spread within layers yielded intralaminar conduction velocities in different layers. Velocities ranged from 74 to 473μm/ms depending on trajectory; interlaminar conduction was 71% faster than intralaminar conduction. Thus, computations within columns are more rapid than between columns. The BC integrates thalamic and intracortical input for functions such as texture discrimination and sensory tuning. Timing differences between intra- and interlaminar PV interneuron activation could impact these functions. Imaging of voltage in PV interneurons reveals differences in signaling dynamics within cortical circuitry. This approach offers a unique opportunity to investigate conduction in populations of axons based on their targeting specificity.

A positively tuned voltage indicator for extended electrical recordings in the brain.

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.

Striatal cholinergic interneuron membrane voltage tracks locomotor rhythms in mice

Rhythmic neural network activity has been broadly linked to behavior. However, it is unclear how membrane potentials of individual neurons track behavioral rhythms, even though many neurons exhibit pace-making properties in isolated brain circuits. To examine whether single-cell voltage rhythmicity is coupled to behavioral rhythms, we focused on delta-frequencies (1–4 Hz) that are known to occur at both the neural network and behavioral levels. We performed membrane voltage imaging of individual striatal neurons simultaneously with network-level local field potential recordings in mice during voluntary movement. We report sustained delta oscillations in the membrane potentials of many striatal neurons, particularly cholinergic interneurons, which organize spikes and network oscillations at beta-frequencies (20–40 Hz) associated with locomotion. Furthermore, the delta-frequency patterned cellular dynamics are coupled to animals’ stepping cycles. Thus, delta-rhythmic cellular dynamics in cholinergic interneurons, known for their autonomous pace-making capabilities, play an important role in regulating network rhythmicity and movement patterning.

Wide-field fluorescence lifetime imaging of neuron spiking and subthreshold activity in vivo.

The development of voltage-sensitive fluorescent probes suggests fluorescence lifetime as a promising readout for electrical activity in biological systems. Existing approaches fail to achieve the speed and sensitivity required for voltage imaging in neuroscience applications. We demonstrated that wide-field electro-optic fluorescence lifetime imaging microscopy (EO-FLIM) allows lifetime imaging at kilohertz frame-acquisition rates, spatially resolving action potential propagation and subthreshold neural activity in live adult Drosophila. Lifetime resolutions of <5 picoseconds at 1 kilohertz were achieved for single-cell voltage recordings. Lifetime readout is limited by photon shot noise, and the method provides strong rejection of motion artifacts and technical noise sources. Recordings revealed local transmembrane depolarizations, two types of spikes with distinct fluorescence lifetimes, and phase locking of spikes to an external mechanical stimulus.

Current Practice in Using Voltage Imaging to Record Fast Neuronal Activity: Successful Examples from Invertebrate to Mammalian Studies.

The optical imaging of neuronal activity with potentiometric probes has been credited with being able to address key questions in neuroscience via the simultaneous recording of many neurons. This technique, which was pioneered 50 years ago, has allowed researchers to study the dynamics of neural activity, from tiny subthreshold synaptic events in the axon and dendrites at the subcellular level to the fluctuation of field potentials and how they spread across large areas of the brain. Initially, synthetic voltage-sensitive dyes (VSDs) were applied directly to brain tissue via staining, but recent advances in transgenic methods now allow the expression of genetically encoded voltage indicators (GEVIs), specifically in selected neuron types. However, voltage imaging is technically difficult and limited by several methodological constraints that determine its applicability in a given type of experiment. The prevalence of this method is far from being comparable to patch clamp voltage recording or similar routine methods in neuroscience research. There are more than twice as many studies on VSDs as there are on GEVIs. As can be seen from the majority of the papers, most of them are either methodological ones or reviews. However, potentiometric imaging is able to address key questions in neuroscience by recording most or many neurons simultaneously, thus providing unique information that cannot be obtained via other methods. Different types of optical voltage indicators have their advantages and limitations, which we focus on in detail. Here, we summarize the experience of the scientific community in the application of voltage imaging and try to evaluate the contribution of this method to neuroscience research.

AI to the rescue of voltage imaging

In a recent issue of Nature Methods, Platisa etal. present an approach for long-term, invivo population voltage imaging with single spike resolution across a local population of 100 neurons.1 Key to this step forward was the combination of a customized high-speed two-photon microscope with an optimized, positive-going, genetically encoded voltage indicator and a tailored machine learning denoising algorithm.

In Memoriam: Lawrence B. Cohen (1939–2023)

In Memoriam: Lawrence B. Cohen (1939–2023)

Cortical Correlates of Psychedelic-Induced Shaking Behavior Revealed by Voltage Imaging.

(1) From mouse to man, shaking behavior (head twitches and/or wet dog shakes) is a reliable readout of psychedelic drug action. Shaking behavior like psychedelia is thought to be mediated by serotonin 2A receptors on cortical pyramidal cells. The involvement of pyramidal cells in psychedelic-induced shaking behavior remains hypothetical, though, as experimental in vivo evidence is limited. (2) Here, we use cell type-specific voltage imaging in awake mice to address this issue. We intersectionally express the genetically encoded voltage indicator VSFP Butterfly 1.2 in layer 2/3 pyramidal neurons. We simultaneously capture cortical hemodynamics and cell type-specific voltage activity while mice display psychedelic shaking behavior. (3) Shaking behavior is preceded by high-frequency oscillations and overlaps with low-frequency oscillations in the motor cortex. Oscillations spectrally mirror the rhythmics of shaking behavior and reflect layer 2/3 pyramidal cell activity complemented by hemodynamics. (4) Our results reveal a clear cortical fingerprint of serotonin-2A-receptor-mediated shaking behavior and open a promising methodological avenue relating a cross-mammalian psychedelic effect to cell-type specific brain dynamics.

Trojan awakener: detecting dormant malicious hardware using laser logic state imaging (extended version)

The threat of (HTs) and their detection is a widely studied field. While the effort for inserting a Trojan into an (ASIC) can be considered relatively high, especially when trusting the chip manufacturer, programmable hardware is vulnerable to Trojan insertion even after the product has been shipped or during usage. At the same time, detecting dormant HTs with small or zero-overhead triggers and payloads on these platforms is still a challenging task, as the Trojan might not get activated during the chip verification using logical testing or physical measurements. In this work, we present a novel Trojan detection approach based on a technique known from (IC) failure analysis, capable of detecting virtually all classes of dormant Trojans. Using (LLSI), we show how supply voltage modulations can awaken inactive Trojans, making them detectable using laser voltage imaging techniques. Therefore, our technique does not require triggering the Trojan. To support our claims, we present three case studies on 28 nm and 20 nm SRAM- and flash-based (FPGAs). We demonstrate how to detect with high confidence small changes in sequential and combinatorial logic as well as in the routing configuration of FPGAs in a non-invasive manner. Finally, we discuss the practical applicability of our approach on dormant analog Trojans in ASICs.

Awakening from anesthesia switches cortical wave modes

Spontaneous cortical activity exhibits diverse spatiotemporal dynamics across brain states, but the organizational principles during state transitions remain unclear. In a recent study combining cortex-wide voltage imaging and neural modeling, Liang and colleagues revealed that global–local competition and long-range connections underlie the emergence of complex cortical wave patterns during awakening from anesthesia.

Voltage Imaging with Engineered Proton-Pumping Rhodopsins: Insights from the Proton Transfer Pathway.

Voltage imaging using genetically encoded voltage indicators (GEVIs) has taken the field of neuroscience by storm in the past decade. Its ability to create subcellular and network level readouts of electrical dynamics depends critically on the kinetics of the response to voltage of the indicator used. Engineered microbial rhodopsins form a GEVI subclass known for their high voltage sensitivity and fast response kinetics. Here we review the essential aspects of microbial rhodopsin photocycles that are critical to understanding the mechanisms of voltage sensitivity in these proteins and link them to insights from efforts to create faster, brighter and more sensitive microbial rhodopsin-based GEVIs.

Multisensory learning binds neurons into a cross-modal memory engram

Associating multiple sensory cues with objects and experience is a fundamental brain process that improves object recognition and memory performance. However, neural mechanisms that bind sensory features during learning and augment memory expression are unknown. Here we demonstrate multisensory appetitive and aversive memory in Drosophila. Combining colours and odours improved memory performance, even when each sensory modality was tested alone. Temporal control of neuronal function revealed visually selective mushroom body Kenyon cells (KCs) to be required for enhancement of both visual and olfactory memory after multisensory training. Voltage imaging in head-fixed flies showed that multisensory learning binds activity between streams of modality-specific KCs so that unimodal sensory input generates a multimodal neuronal response. Binding occurs between regions of the olfactory and visual KC axons, which receive valence-relevant dopaminergic reinforcement, and is propagated downstream. Dopamine locally releases GABAergic inhibition to permit specific microcircuits within KC-spanning serotonergic neurons to function as an excitatory bridge between the previously ‘modality-selective’ KC streams. Cross-modal binding thereby expands the KCs representing the memory engram for each modality into those representing the other. This broadening of the engram improves memory performance after multisensory learning and permits a single sensory feature to retrieve the memory of the multimodal experience.

Review of clinical applications and challenges with surface-guided radiation therapy.

To evaluate the use of this new technique, surface-guided radiotherapy (SGRT), for patient setup and motion management in various cancers. Data was collected from 533 patients, who received treatment in our hospital for various malignancies using SGRT from October 2019 to April 2021. We studied patient setup, interfraction position, and patient position during the breath-hold (BH) technique. The main advantage of SGRT is that, it is completely non-invasive and uses visible light to compare the patient's skin surface in the treatment room and planned treatment position. In this analysis, Monaco 5.51.10 (Elekta) treatment planning system, Versa HD Linear Accelerator, and AlignRT 6.2 (Vision RT) SGRT system were used. With SGRT, treatment setup time can be reduced with more precision and techniques like Deep inspiration breathhold (DIBH) can be done with very good compliance. SGRT has shown improved accuracy in patient setup compared to conventional laser setup. The daily kilo voltage imaging frequency can be reduced; it helps in reducing additional radiation exposure due to imaging. SGRT has demonstrated reproducibility with adequate accuracy in BH treatments in DIBH for breast and SBRT.

Simulations of active zone structure and function at mammalian NMJs predict that loss of calcium channels alone is not sufficient to replicate LEMS effects.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune-mediated neuromuscular disease thought to be caused by autoantibodies against P/Q-type voltage-gated calcium channels (VGCCs), which attack and reduce the number of VGCCs within transmitter release sites (active zones; AZs) at the neuromuscular junction (NMJ), resulting in neuromuscular weakness. However, patients with LEMS also have antibodies to other neuronal proteins, and about 15% of patients with LEMS are seronegative for antibodies against VGCCs. We hypothesized that a reduction in the number of P/Q-type VGCCs alone is not sufficient to explain LEMS effects on transmitter release. Here, we used a computational model to study a variety of LEMS-mediated effects on AZ organization and transmitter release constrained by electron microscopic, pharmacological, immunohistochemical, voltage imaging, and electrophysiological observations. We show that models of healthy AZs can be modified to predict the transmitter release and short-term facilitation characteristics of LEMS and that in addition to a decrease in the number of AZ VGCCs, disruption in the organization of AZ proteins, a reduction in AZ number, a reduction in the amount of synaptotagmin, and the compensatory expression of L-type channels outside the remaining AZs are important contributors to LEMS-mediated effects on transmitter release. Furthermore, our models predict that antibody-mediated removal of synaptotagmin in combination with disruption in AZ organization alone could mimic LEMS effects without the removal of VGCCs (a seronegative model). Overall, our results suggest that LEMS pathophysiology may be caused by a collection of pathological alterations to AZs at the NMJ, rather than by a simple loss of VGCCs.NEW & NOTEWORTHY We used a computational model of the active zone (AZ) in the mammalian neuromuscular junction to investigate Lambert-Eaton myasthenic syndrome (LEMS) pathophysiology. This model suggests that disruptions in presynaptic active zone organization and protein content (particularly synaptotagmin), beyond the simple removal of presynaptic calcium channels, play an important role in LEMS pathophysiology.

Accuracy of abdominal organ motion estimation in radiotherapy using the right hemidiaphragm top as a surrogate during prolonged breath-holds quantified with MRI.

Respiratory motion presents a challenge in radiotherapy of thoracic and upper abdominal tumors. Techniques to account for respiratory motion include tracking. Using magnetic resonance imaging (MRI) guided radiotherapy systems, tumors can be tracked continuously. Using conventional linear accelerators, tracking of lung tumors is possible by determining tumor motion on kilo voltage (kV) imaging. But tracking of abdominal tumors with kV imaging is hampered by limited contrast. Therefore, surrogates for the tumor are used. One of the possible surrogates is the diaphragm. However, there is no universal method for establishing the error when using a surrogate and there are particular challenges in establishing such errors during free breathing (FB). Prolonged breath-holding might address these challenges. The aim of this study was to quantify the error when using the right hemidiaphragm top (RHT) as surrogate for abdominal organ motion during prolonged breath-holds (PBH) for possible application in radiation treatments. Fifteen healthy volunteers were trained to perform PBHs in two subsequent magnetic resonance imaging (MRI) sessions (PBH-MRI1 and PBH-MRI2). From each MRI acquisition, we selected seven images (dynamics) to determine organ displacement during PBH by using deformable image registration (DIR). On the first dynamic, the RHT, right and left hemidiaphragm, liver, spleen and right and left kidney were segmented. We used the deformation vector fields (DVF), generated by DIR, to determine the displacement of each organ between two dynamics in inferior-superior (IS), anterior-posterior (AP), left-right (LR) direction and we calculated the 3D vector magnitude (|d|). The displacements of the RHT, both hemidiaphragms and the abdominal organs were compared using a linear fit to determine the correlation (R2 of the fit) and the displacement ratio (DR, slope of the fit) between displacements of the RHT and each organ. We quantified the median difference between the DRs of PBH-MRI1 and PBH-MRI2 for each organ. Additionally, we estimated organ displacement in the second PBH by applying the DR from the first PBH to the displacement of the RHT measured during the second PBH. We compared the estimated organ displacement to the measured organ displacement during the second PBH. The difference between the two values was defined as the estimation error of using the RHT as a surrogate and assuming a constant DR over MRI sessions. The linear relationships were confirmed by the high R2 values of the linear fit between the displacements of the RHT and the abdominal organs (R2 > 0.96) in the IS and AP direction and |d|, and high to moderate correlations in the LR direction (0.93 > R2 > 0.64). The median DR difference between PBH-MRI1 and PBH-MRI2 varied between 0.13 to 0.31 for all organs. The median estimation error of the RHT as a surrogate varied between 0.4 and 0.8 mm/min for all organs. The RHT could serve as an accurate surrogate for abdominal organ motion during radiation treatments, e.g. in tracking, provided the error of the RHT as motion surrogate is taken into account in the margins. The study was registered in the Netherlands Trial Register (NL7603). This article is protected by copyright. All rights reserved.