DeepVID v2: self-supervised denoising with decoupled spatiotemporal enhancement for low-photon voltage imaging.

Significance: Voltage imaging is a powerful tool for studying the dynamics of neuronal activities in the brain. However, voltage imaging data are fundamentally corrupted by severe Poisson noise in the low-photon regime, which hinders the accurate extraction of neuronal activities. Self-supervised deep learning denoising methods have shown great potential in addressing the challenges in low-photon voltage imaging without the need for ground-truth but usually suffer from the trade-off between spatial and temporal performances.

Plateau depolarizations in spontaneously active neurons detected by calcium or voltage imaging.

In calcium imaging studies, Ca transients are commonly interpreted as neuronal action potentials (APs). However, our findings demonstrate that robust optical Ca transients primarily stem from complex "AP-Plateaus", while simple APs lacking underlying depolarization envelopes produce much weaker photonic signatures. Under challenging in vivo conditions, these "AP-Plateaus" are likely to surpass noise levels, thus dominating the Ca recordings.

DeepVID v2: Self-Supervised Denoising with Decoupled Spatiotemporal Enhancement for Low-Photon Voltage Imaging.

Significance: Voltage imaging is a powerful tool for studying the dynamics of neuronal activities in the brain. However, voltage imaging data are fundamentally corrupted by severe Poisson noise in the low-photon regime, which hinders the accurate extraction of neuronal activities. Self-supervised deep learning denoising methods have shown great potential in addressing the challenges in low-photon voltage imaging without the need for ground truth, but usually suffer from the tradeoff between spatial and temporal performance.

High-speed low-light in vivo two-photon voltage imaging of large neuronal populations.

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.

Voltage imaging in the olfactory bulb using transgenic mouse lines expressing the genetically encoded voltage indicator ArcLight.

Genetically encoded voltage indicators (GEVIs) allow optical recordings of membrane potential changes in defined cell populations. Transgenic reporter animals that facilitate precise and repeatable targeting with high expression levels would further the use of GEVIs in the in vivo mammalian brain. However, the literature on developing and applying transgenic mouse lines as vehicles for GEVI expression is limited.

Genetically encoded fluorescent voltage indicators: are we there yet?

In order to understand how brain activity produces adaptive behavior we need large-scale, high-resolution recordings of neuronal activity. Fluorescent genetically encoded voltage indicators (GEVIs) offer the potential for these recordings to be performed chronically from targeted cells in a minimally invasive manner. As the number of GEVIs successfully tested for in vivo use grows, so has the number of open questions regarding the improvements that would facilitate broad adoption of this technology that surpasses mere 'proof of principle' studies.

Directed Evolution of Key Residues in Fluorescent Protein Inverses the Polarity of Voltage Sensitivity in the Genetically Encoded Indicator ArcLight.

Genetically encoded calcium indicators (GECIs) produce unprecedentedly large signals that have enabled routine optical recording of single neuron activity in vivo in rodent brain. Genetically encoded voltage indicators (GEVIs) offer a more direct measure of neuronal electrical status, however the signal-to-noise characteristics and signal polarity of the probes developed to date have precluded routine use in vivo. We applied directed evolution to target modulable areas of the fluorescent protein in GEVI ArcLight to create the first GFP-based GEVI (Marina) that exhibits a ΔF/ΔV with a positive slope relationship.

A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices.

Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators.

Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment.

We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs) from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs). Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp.

Fluorescent protein voltage probes derived from ArcLight that respond to membrane voltage changes with fast kinetics.

We previously reported the discovery of a fluorescent protein voltage probe, ArcLight, and its derivatives that exhibit large changes in fluorescence intensity in response to changes of plasma membrane voltage. ArcLight allows the reliable detection of single action potentials and sub-threshold activities in individual neurons and dendrites. The response kinetics of ArcLight (τ1-on ~10 ms, τ2-on ~ 50 ms) are comparable with most published genetically-encoded voltage probes.

Genetically targeted optical electrophysiology in intact neural circuits.

Nervous systems process information by integrating the electrical activity of neurons in complex networks. This motivates the long-standing interest in using optical methods to simultaneously monitor the membrane potential of multiple genetically targeted neurons via expression of genetically encoded fluorescent voltage indicators (GEVIs) in intact neural circuits. No currently available GEVIs have demonstrated robust signals in intact brain tissue that enable reliable recording of individual electrical events simultaneously in multiple neurons.

A fluorescent, genetically-encoded voltage probe capable of resolving action potentials.

There is a pressing need in neuroscience for genetically-encoded, fluorescent voltage probes that can be targeted to specific neurons and circuits to allow study of neural activity using fluorescent imaging. We created 90 constructs in which the voltage sensing portion (S1-S4) of Ciona intestinalis voltage sensitive phosphatase (CiVSP) was fused to circularly permuted eGFP. This led to ElectricPk, a probe that is an order of magnitude faster (taus ~1-2 ms) than any currently published fluorescent protein-based voltage probe.

Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe.

Monitoring neuronal electrical activity using fluorescent protein-based voltage sensors has been limited by small response magnitudes and slow kinetics of existing probes. Here we report the development of a fluorescent protein voltage sensor, named ArcLight, and derivative probes that exhibit large changes in fluorescence intensity in response to voltage changes. ArcLight consists of the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase and super ecliptic pHluorin that carries the point mutation A227D.

Genetically encoded fluorescent voltage sensors using the voltage-sensing domain of Nematostella and Danio phosphatases exhibit fast kinetics.

A substantial increase in the speed of the optical response of genetically encoded fluorescent protein voltage sensors (FP voltage sensors) was achieved by using the voltage-sensing phosphatase genes of Nematostella vectensis and Danio rerio. A potential N. vectensis voltage-sensing phosphatase was identified in silico.

Herbicide phosphinothricin causes direct stimulation hormesis.

Herbicide phosphinothricin (PPT) inhibits glutamine synthetase (GS), a key enzyme in nitrogen assimilation, thus causing ammonia accumulation, glutamine depletion and eventually plant death. However, the growth response of Lotus corniculatus L. plants immersed in solutions with a broad range of PPT concentrations is biphasic, with pronounced stimulating effect on biomass production at concentrations ≤ 50 μM and growth inhibition at higher concentrations.

Induction of peroxidases and superoxide dismutases in transformed embryogenic calli of alfalfa (Medicago sativa L.).

Peroxidase (POD) and superoxide dismutase (SOD) enzyme activities were analyzed in non-regenerative transformed embryogenic lines of alfalfa (Medicago sativa L.) carrying wound-inducible oryzacystatin I (OC-I), wound-inducible oryzacystatin I antisense (OC-Ias), or hygromycin phosphotransferase (hpt) genes. All of the transformed lines analyzed had elevated levels of all POD isoforms.