Biology subjects

Pieribone, V. A.

Publications and source records attributed to Pieribone, V. A..

7 recordsLinked to original sources

bioRxiv2024

Velamins: the first green-light emitting class of wild-type Ca2+-regulated photoproteins isolated from the ctenophore Velamen parallelum

Ca2+-regulated photoproteins (CaPhs) consist of single-chain globular proteins to which coelenterazine, a widely distributed marine luminogenic substrate (the luciferin), binds along with molecular oxygen, producing a stable peroxide. Upon Ca2+ addition, CaPhs undergo conformational changes leading to the cyclization of the peroxide and the formation of a high-energy intermediate. Subsequently, its decomposition yields coelenteramide in an excited state and results in the emission of a flash of light. To date, all CaPhs reported produce blue light ({lambda}max 465-495 nm). Here, we report the cloning and functional characterization of a novel class of wild-type CaPhs capable of emitting green light: velamins, isolated from the bioluminescent ctenophore Velamen parallelum. Ten unique photoprotein-like sequences were recovered and grouped in three main clusters. Representative sequences were cloned, expressed, purified, and regenerated into the active His-tagged -, {beta}-, and {gamma}-velamins. Upon injection of a calcium-containing buffer into the velamin, a flash of green light ({lambda}max 500-508 nm) was observed across pH values ranging from 7 to 9. Whilst -velamin isoforms exhibited the highest light emission activity, {beta}- and {gamma}-velamins were found to be more thermostable at higher temperatures. Velamins are the only known wild-type Ca2+-regulated photoproteins that exhibit the longest wavelength in light emission, making them a promising model for studying spectral modulation. As a result, velamins hold potential for enhancing the sensitivity of signal detection in analytical systems, particularly when dealing with complex biological matrices.

biochemistry↗

bioRxiv2024

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

SignificanceVoltage 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. AimWe present DeepVID v2, a novel self-supervised denoising framework with decoupled spatial and temporal enhancement capability to significantly augment low-photon voltage imaging. ApproachDeepVID v2 is built on our original DeepVID framework,1,2 which performs frame-based denoising by utilizing a sequence of frames around the central frame targeted for denoising to leverage temporal information and ensure consistency. The network further integrates multiple blind pixels in the central frame to enrich the learning of local spatial information. Additionally, DeepVID v2 introduces a new edge extraction branch to capture fine structural details in order to learn high spatial resolution information. ResultsWe demonstrate that DeepVID v2 is able to overcome the tradeoff between spatial and temporal performance, and achieve superior denoising capability in resolving both high-resolution spatial structures and rapid temporal neuronal activities. We further show that DeepVID v2 is able to generalize to different imaging conditions, including time-series measurements with various signal-to-noise ratios (SNRs) and in extreme low-photon conditions. ConclusionsOur results underscore DeepVID v2 as a promising tool for enhancing voltage imaging. This framework has the potential to generalize to other low-photon imaging modalities and greatly facilitate the study of neuronal activities in the brain.

neuroscience↗

bioRxiv2023

Subdural CMOS optical probe (SCOPe) for bidirectional neural interfacing

Optical neurotechnologies use light to interface with neurons and can monitor and manipulate neural activity with high spatial-temporal precision over large cortical extents. While there has been significant progress in miniaturizing microscope for head-mounted configurations, these existing devices are still very bulky and could never be fully implanted. Any viable translation of these technologies to human use will require a much more noninvasive, fully implantable form factor. Here, we leverage advances in microelectronics and heterogeneous optoelectronic packaging to develop a transformative, ultrathin, miniaturized device for bidirectional optical stimulation and recording: the subdural CMOS Optical Probe (SCOPe). By being thin enough to lie entirely within the subdural space of the primate brain, SCOPe defines a path for the eventual human translation of a new generation of brain-machine interfaces based on light.

neuroscience↗

bioRxiv2021

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 which reduces fluorescence detection to near shot noise levels. High-photon flux excitation can overcome photon-limited shot noise but photo-bleaching and photo-damage restricts the number and duration of simultaneously imaged neurons. We investigated an alternative approach aimed at low two-photon flux, voltage imaging below the shot noise limit. This framework involved developing: a positive-going voltage indicator with improved spike detection (SpikeyGi); an ultra-fast two-photon microscope for kilohertz frame-rate imaging across a 0.4x0.4mm2 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 one hundred densely-labeled neurons over one hour in awake behaving mice. This demonstrates a scalable approach for voltage imaging across increasing neuronal populations.

neuroscience↗

bioRxiv2021

Dual polarity voltage imaging reveals subthreshold dynamics and concurrent spiking patterns of multiple neuron-types

Genetically encoded fluorescent voltage indicators are ideally suited to reveal the millisecond-scale interactions among and between distinct, targeted cell populations. However, current indicator families lack the requisite sensitivity for in vivo multipopulation imaging. We describe high-performance green and red sensors, Ace-mNeon2 and VARNAM2, and their reverse response-polarity variants, pAce and pAceR. Our indicators enable 0.4-1 kHz voltage recordings from >50 neurons per field-of-view in awake mice and [~]30-min continuous imaging in flies. Using dual-polarity multiplexed imaging, we uncovered behavioral state-dependent interactions between distinct neocortical subclasses, as well as contributions to hippocampal field potentials from non-overlapping projection neuronal ensembles. By combining three mutually compatible indicators, we demonstrate concurrent triple-population voltage imaging. Our approach will empower investigations of the dynamic interplay between neuronal subclasses at single-spike resolution. One Sentence SummaryA new suite of voltage sensors enables simultaneous cellular-resolution activity imaging from multiple, targeted neuron-types in awake animals.

neuroscience↗

bioRxiv2020

Voltage imaging using transgenic mouse lines expressing the GEVI ArcLight in two olfactory cell types

Genetically encoded voltage indicators (GEVIs) allow for cell-specific optical recordings of membrane potential changes in defined cell populations. One tool that would further their use in the in vivo mammalian brain is transgenic reporter animals that facilitate precise and repeatable targeting with high expression levels. The present literature on the development and use of transgenic mouse lines as vehicles for GEVI expression is limited. Here we report the first in vivo experiments using a transgenic reporter mouse for the GEVI ArcLight (Ai86(TITL-ArcLight)), which utilizes a Cre/tTA dependent expression system (TIGRE 1.0). Following pairing to appropriate Cre- and tTA transgenic mice, we report two mouse lines with ArcLight expression restricted to olfactory sensory neurons (OMP-ArcLight), and a subpopulation of interneurons that include periglomerular and granule cells (Emx1-ArcLight) in the olfactory bulb (OB). The ArcLight expression in these lines was sufficient for in vivo imaging of odorant responses in single trials. Odor responses were measured in the OB using epifluorescence and 2-photon imaging. The voltage responses were odor-specific and concentration-dependent, and confirmed earlier conclusions from calcium measurements. This study shows that the ArcLight Ai86(TITL-ArcLight) transgenic line is a flexible genetic tool that can be used to record neuronal electrical activity of a variety of cell types with a signal-to-noise ratio that is comparable to previous reports using viral transduction.

neuroscience↗

bioRxiv2020

Different categories of fluorescent proteins result in GEVIs with similar characteristics

The latest generation of genetically encoded voltage indicators (GEVIs) is significantly advancing our ability to study electrical activity from large numbers of identified neurons. The further refinement of the technology will contribute to our understanding of behavior-evoked information perception, transfer and processing on a cellular level across brain regions. The development of GEVIs relies on synthetic biology which includes rational and random modifications of indicator sequence. One strategy in GEVI design is based on creating chimeras between voltage sensitive protein domains (VSDs) and fluorescent proteins (FPs). However, in this design scenario, the mechanistic details of voltage-induced fluorescence change that would inform rational design and improvements of GEVIs are still largely missing. Here we preformed a systematic study of how nature of the FP and altering the insertion site affects the characteristics of Ciona intestinalis voltage-sensitive phosphatase-based GEVIs. Surprisingly, we found that regardless of vast difference in phylogenesis, biochemical properties, fluorophore structure, sequence and excitation/emission spectra between FPs, the resulting GEVIs exhibit virtually identical decrease in fluorescence intensity in response to depolarization. These results stand in strong contrast to studies demonstrating that small numbers of targeted mutations in the FP sequence cause dramatic changes in both signal size and polarity.

neuroscience↗