Biology subjects

Miller, E.

Publications and source records attributed to Miller, E..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Long-term, super-resolution HIDE imaging of the inner mitochondrial membrane in live cells with a cell-permeant lipid probe

The densely packed inner mitochondrial membrane (IMM) is vital for bioenergy generation and its dynamics control mitochondrial health and cellular homeostasis. IMM structure is complex, however, and imaging its dynamics with high temporal and spatial resolution is complicated by the photosensitivity of IMM-resident enzymes. Here we describe the cell-permeant, lipid-like acridine orange derivative MAO-N3 and use it to assemble high-density, environmentally sensitive (HIDE) probes that selectively label and image the IMM in live cells. MAO-N3 pairs with multiple SPAAC-reactive fluorophores to support HIDE imaging via confocal, Structured Illumination, Single Molecule Localization, and Stimulated Emission Depletion microscopy, all with significantly improved resistance against photobleaching. The HIDE probes generated using MAO-N3 require no genetic manipulations, are non-toxic in model cell lines and primary cardiomyocytes, even under conditions that amplify the effects of mitochondrial toxins, and visualize the IMM for up to 12.5 hours with unprecedented spatial and temporal resolution.

cell biology↗

bioRxiv2022

Longitudinal in vivo monitoring of axonal integrity after brain injury

Traumatic brain injury-induced axonal degeneration leads to acute and chronic neuropsychiatric impairment, neuronal death, and accelerated neurodegenerative diseases of aging, including Alzheimers and Parkinsons diseases. Thus, there is much interest in developing treatments that protect axons after injury. For this endeavor, extended comprehensive evaluation of axonal integrity in experimental systems is required to evaluate the efficacy of putative interventions in preclinical models. However, traditional histological tissue proccessing techniques are logistically prohibitive for assessments of long-term pathology. Here, we report a new method of longitudinally monitoring the functional activity of thalamocortical axons before and after injury in vivo in the same animal over an extended period of time. Specifically, we expressed an axonal-targeting genetically-encoded calcium indicator in the mouse dorsolateral geniculate nucleus and then recorded axonal activity patterns in the visual cortex in response to visual stimulation. We demonstrate the utility of this method for assessing in vivo aberrant axonal activity patterns after traumatic brain injury, as well as for evaluating the therapuetic efficacy of the neuroprotective P7C3-A20 pharmacologic agent in vivo. We found that P7C3-A20 treatment minimized most, but not all, of the pathological changes in axonal activity patterns after traumatic brain injury.

neuroscience↗

bioRxiv2022

High-frame-rate analysis of knee cartilage deformation by spiral dualMRI and relaxation mapping

PurposeDaily activities including walking impose high frequency cyclic forces on cartilage and repetitive compressive deformation. Analyzing cartilage deformation during walking would provide spatial maps of displacement, strain, and enable viscoelastic characterization, which may serve as imaging biomarkers for early cartilage degeneration when the damage is still reversible. However, the time-dependent biomechanics of cartilage is not well described, and how defects in the joint impact the viscoelastic response is unclear. MethodsWe used spiral acquisition with displacement encoding MRI to quantify displacement and strain maps at a high frame rate (40 ms; 25 frames/sec) in tibiofemoral joints. We also employed relaxometry methods (T1, T1{rho}, T2, T2*) on the cartilage. ResultsNormal and shear strains were concentrated on the tibiofemoral contact area during loading, and the defected joint exhibited larger compressive strains. We also determined a positive correlation between the change of T1{rho} in cartilage after cyclic loading and increased compressive strain on the defected joint. Viscoelastic behavior was quantified by the time-dependent displacement, where the damaged joint showed increased creep behavior compared to the intact. ConclusionsOur results indicate that spiral scanning with displacement encoding can quantitatively differentiate the damaged from intact joint using the strain and creep response. The viscoelastic response identified with this methodology could serve as biomarkers to detect defects in joints in vivo and facilitate the early diagnosis of joint diseases such as osteoarthritis.

bioengineering↗

bioRxiv2021

Voltage imaging in Drosophila using a hybrid chemical-genetic rhodamine voltage reporter

We combine a chemically-synthesized, voltage-sensitive fluorophore with a genetically encoded, self-labeling enzyme to enable voltage imaging in Drosophila melanogaster. Previously, we showed that a rhodamine voltage reporter (RhoVR) combined with the HaloTag self-labeling enzyme could be used to monitor membrane potential changes from mammalian neurons in culture and brain slice. Here, we apply this hybrid RhoVR-Halo approach in vivo to achieve selective neuron labeling in intact fly brains. We generate a Drosophila UAS-HaloTag reporter line in which the HaloTag enzyme is expressed on the surface of cells. We validate the voltage sensitivity of this new construct in cell culture before driving expression of HaloTag in specific brain neurons in flies. We show that selective labeling of synapses, cells, and brain regions can be achieved with RhoVR-Halo in either larval neuromuscular junction (NMJ) or in whole adult brains. Finally, we validate the voltage sensitivity of RhoVR-Halo in fly tissue via dual-electrode/imaging at the NMJ, show the efficacy of this approach for measuring synaptic excitatory post-synaptic potentials (EPSPs) in muscle cells, and perform voltage imaging of carbachol-evoked depolarization and osmolarity-evoked hyperpolarization in projection neurons and in interoceptive subesophageal zone neurons in fly brain explants following in vivo labeling. We envision the turn-on response to depolarizations, fast response kinetics, and two-photon compatibility of chemical indicators, coupled with the cellular and synaptic specificity of genetically-encoded enzymes, will make RhoVR-Halo a powerful complement to neurobiological imaging in Drosophila. Significance StatementVoltage imaging is a powerful method for interrogating neurobiology. Chemical indicators possess fast response kinetics, turn-on responses to membrane depolarization, and can be compatible with two-photon excitation. However, selective cell labeling in intact tissues and in vivo remains a challenge for completely synthetic fluorophores. Here, we show that a chemical - genetic hybrid approach in Drosophila enables cell-specific staining in vivo and voltage imaging in whole-brain explants and at neuromuscular junction synapses.

neuroscience↗

bioRxiv2021

11β-HSD1 inhibition does not affect murine tumour angiogenesis but may exert a selective effect on tumour growth by modulating inflammation and fibrosis

Glucocorticoids inhibit angiogenesis by activating the glucocorticoid receptor. Inhibition of the glucocorticoid-activating enzyme 11{beta}-hydroxysteroid dehydrogenase type 1 (11{beta}-HSD1) reduces tissue-specific glucocorticoid action and promotes angiogenesis in murine models of myocardial infarction. Angiogenesis is important in the growth of some solid tumours. This study used murine models of squamous cell carcinoma (SCC) and pancreatic ductal adenocarcinoma (PDAC) to test the hypothesis that 11{beta}-HSD1 inhibition promotes angiogenesis and subsequent tumour growth. SCC or PDAC cells were injected into female FVB/N or C57BL6/J mice fed either standard diet, or diet containing the 11{beta}-HSD1 inhibitor UE2316. SCC tumours grew more rapidly in UE2316-treated mice, reaching a larger (P<0.01) final volume (0.158 {+/-} 0.037 cm3) than in control mice (0.051 {+/-} 0.007 cm3). However, PDAC tumour growth was unaffected. Immunofluorescent analysis of SCC tumours did not show differences in vessel density (CD31/alpha-smooth muscle actin) or cell proliferation (Ki67) after 11{beta}-HSD1 inhibition, and immunohistochemistry of SCC tumours did not show changes in inflammatory cell (CD3- or F4/80-positive) infiltration. In culture, the growth/viability (assessed by live cell imaging) of SCC cells was not affected by UE2316 or corticosterone. Second Harmonic Generation microscopy showed that UE2316 reduced Type I collagen (P<0.001), whilst RNA-sequencing revealed that multiple factors involved in the innate immune/inflammatory response were reduced in UE2316-treated SCC tumours. 11{beta}-HSD1 inhibition increases SCC tumour growth, likely via suppression of inflammatory/immune cell signalling and extracellular matrix deposition, but does not promote tumour angiogenesis or growth of all solid tumours.

cancer biology↗

bioRxiv2021

OpenNeuro: An open resource for sharing of neuroimaging data

The sharing of research data is essential to ensure reproducibility and maximize the impact of public investments in scientific research. Here we describe OpenNeuro, a BRAIN Initiative data archive that provides the ability to openly share data from a broad range of brain imaging data types following the FAIR principles for data sharing. We highlight the importance of the Brain Imaging Data Structure (BIDS) standard for enabling effective curation, sharing, and reuse of data. The archive presently shares more than 600 datasets including data from more than 20,000 participants, comprising multiple species and measurement modalities and a broad range of phenotypes. The impact of the shared data is evident in a growing number of published reuses, currently totalling more than 150 publications. We conclude by describing plans for future development and integration with other ongoing open science efforts.

neuroscience↗

bioRxiv2021

Mutation rate dynamics reflect ecological changes in an emerging zoonotic pathogen

While mutation is often deleterious, it can also be adaptive. Mutation rates vary both within and between bacterial species, and understanding what drives this variation is essential for understanding the evolutionary dynamics of bacterial populations. In this study we investigate two factors that are predicted to influence the trade-off between the costs and benefits of mutation: ecology and genome size. To investigate the relationship between these factors and mutation rate we conducted mutation accumulation experiments on eight strains of the emerging zoonotic pathogen Streptococcus suis. Natural variation within this species allows us to compare tonsil carriage and invasive disease isolates, from both more and less pathogenic populations, with a wide range of genome sizes. We find that invasive disease isolates have repeatedly evolved mutation rates that are higher than those of closely related carriage isolates, regardless of variation in genome size. Independent of this variation in overall rate, we also observe a stronger bias towards G/C to A/T mutations in isolates from more pathogenic populations, whose genomes tend to be smaller and more AT-rich. Our results suggest that ecology is a stronger correlate of mutation rate than genome size over these timescales, and that transitions to invasive disease are consistently accompanied by rapid increases in mutation rate. These results shed light on the impact ecology can have on the adaptive potential of bacterial pathogens.

evolutionary biology↗

bioRxiv2021

Optical Estimation of Absolute Membrane Potential Using One- and Two-Photon Fluorescence Lifetime Imaging Microscopy

BackgroundMembrane potential (Vmem) exerts physiological influence across a wide range of time and space scales. To study Vmem in these diverse contexts, it is essential to accurately record absolute values of Vmem, rather than solely relative measurements. Materials & MethodsWe use fluorescence lifetime imaging of a small molecule voltage sensitive dye (VF2.1.Cl) to estimate mV values of absolute membrane potential. ResultsWe test the consistency of VF2.1.Cl lifetime measurements performed on different single photon counting instruments and find that they are in striking agreement (differences of <0.5 ps/mV in the slope and <50 ps in the y-intercept). We also demonstrate that VF2.1.Cl lifetime reports absolute Vmem under two-photon (2P) illumination with better than 20 mV of Vmem resolution, a nearly 10-fold improvement over other lifetime-based methods. ConclusionsWe demonstrate that VF-FLIM is a robust and portable metric for Vmem across imaging platforms and under both one-photon and two-photon illumination. This work is a critical foundation for application of VF-FLIM to record absolute membrane potential signals in thick tissue.

biophysics↗

bioRxiv2020

In Vitro Safety Clinical Trial of the Cardiac Liability of Hydroxychloroquine and Azithromycin as COVID19 Polytherapy

Despite global efforts, there are no effective FDA-approved medicines for the treatment of SARS-CoV-2 infection. Potential therapeutics focus on repurposed drugs, some with cardiac liabilities. Here we report on a preclinical drug screening platform, a cardiac microphysiological system (MPS), to assess cardiotoxicity associated with hydroxychloroquine (HCQ) and azithromycin (AZM) polytherapy in a mock clinical trial. The MPS contained human heart muscle derived from patient-specific induced pluripotent stem cells. The effect of drug response was measured using outputs that correlate with clinical measurements such as QT interval (action potential duration) and drug-biomarker pairing. Chronic exposure to HCQ alone elicited early afterdepolarizations (EADs) and increased QT interval from day 6 onwards. AZM alone elicited an increase in QT interval from day 7 onwards and arrhythmias were observed at days 8 and 10. Monotherapy results closely mimicked clinical trial outcomes. Upon chronic exposure to HCQ and AZM polytherapy, we observed an increase in QT interval on days 4-8.. Interestingly, a decrease in arrhythmias and instabilities was observed in polytherapy relative to monotherapy, in concordance with published clinical trials. Furthermore, biomarkers, most of them measurable in patients serum, were identified for negative effects of single drug or polytherapy on tissue contractile function, morphology, and antioxidant protection. The cardiac MPS can predict clinical arrhythmias associated with QT prolongation and rhythm instabilities. This high content system can help clinicians design their trials, rapidly project cardiac outcomes, and define new monitoring biomarkers to accelerate access of patients to safe COVID-19 therapeutics.

bioengineering↗

bioRxiv2020

The Wood Image Analysis and Dataset (WIAD): open-access visual analysis tools to advance the ecological data revolution

O_LIEcological data is collected and shared at an increasingly rapid pace, but it is often shared in inconsistent and untraceable processed forms. Images of wood contain a wealth of information such as colours and textures but are most commonly reduced to ring-width measurements before they can be shared in various common file formats. Archiving digital images of wood samples in libraries, that have been developed for ecological analysis and are publicly available, remains the exception. C_LIO_LIWe developed the Wood Image Analysis and Dataset (WIAD) an open-source application including a web-interface to integrate basic visual analysis of wood samples, such as increment cores, thin sections or x-ray films, basic data processing, and archiving of the images and derived data to facilitate transparency and reproducibility in studies using visual characteristics of wood. C_LIO_LIWIAD provides user-friendly tools to manipulate images of wood samples, mark and measure wood characteristics such as growth increments, density fluctuations, early- and latewood widths, and fire scars, and to visualise, process, and archive images, metadata, and the derived data. C_LIO_LIWIAD constitutes a step towards the reproducible automation of tree-ring analysis, while establishing an open-source foundation to create improved community developed repositories which would enable novel ecological studies harnessing the wealth of existing visual data. C_LI

plant biology↗

bioRxiv2020

Optical spike detection and connectivity analysis with a far-red voltage-sensitive fluorophore reveals changes to network connectivity in development and disease

The ability to optically record dynamics of neuronal membrane potential promises to revolutionize our understanding of neurobiology. In this study, we show that the far-red voltage sensitive fluorophore, Berkeley Red Sensor of Transmembrane potential -1, or BeRST 1, can be used to monitor neuronal membrane potential changes across dozens of neurons at a sampling rate of 500 Hz. Notably, voltage imaging with BeRST 1 can be implemented with affordable, commercially available illumination sources, optics, and detectors. BeRST 1 is well-tolerated in cultures of rat hippocampal neurons and provides exceptional optical recording fidelity, as judged by dual fluorescence imaging and patch-clamp electrophysiology. We developed a semi-automated spike-picking program to reduce user bias when calling action potentials and used this in conjunction with BeRST 1 to develop an optical spike and connectivity analysis workflow (OSCA) for high-throughput dissection of neuronal activity dynamics in development and disease. The high temporal resolution of BeRST 1 enables dissection of firing rate changes in response to acute, pharmacological interventions with commonly used inhibitors like gabazine and picrotoxin. Over longer periods of time, BeRST 1 also tracks chronic perturbations to neurons exposed to amyloid beta (A{beta}1-42), revealing modest changes to spiking frequency but profound changes to overall network connectivity. Finally, we use OSCA to track changes in neuronal connectivity during development, providing a functional readout of network assembly. We envision that use of BeRST 1 and OSCA described here will be of use to the broad neuroscience community. Significance StatementOptical methods to visualize membrane potential dynamics provide a powerful complement to Ca2+ imaging, patch clamp electrophysiology, and multi-electrode array recordings. However, modern voltage imaging strategies often require complicated optics, custom-built microscopes, or genetic manipulations that are impractical outside of a subset of model organisms. Here, we describe the use of Berkeley Red Sensor of Transmembrane potential, or BeRST 1, a far-red voltage-sensitive fluorophore that can directly visualize membrane potential changes with millisecond resolution across dozens of neurons. Using only commercially available components, voltage imaging with BeRST 1 reveals profound changes in neuronal connectivity during development, exposes changes to firing rate during acute pharmacological perturbation, and illuminates substantial increases in network connectivity in response to chronic exposure to amyloid beta.

neuroscience↗

bioRxiv2020

Imaging voltage in complete neuronal networks within patterned microislands reveals preferential wiring of excitatory hippocampal neurons

Voltage imaging with fluorescent dyes affords the opportunity to map neuronal activity in both time and space. One limitation to imaging is the inability to image complete neuronal networks: some fraction of cells remains outside of the observation window. Here, we combine voltage imaging, post hoc immunocytochemistry, and patterned microisland hippocampal culture to provide imaging of complete neuronal networks. The patterned microislands completely fill the field of view of our high-speed (500 Hz) camera, enabling reconstruction of the spiking patterns of every single neuron in the network. Cultures raised on microislands develop similarly to neurons grown on coverslips and display similar composition of inhibitory and excitatory cell types. The principal excitatory cell types (CA1, CA3, and dentate granule cells, or DGC) are also present in similar proportions in both preparations. We calculate the likelihood that action potential firing in one neuron to trigger action potential firing in a downstream neuron in a spontaneously active network to construct a functional connection map of these neuronal ensembles. Importantly, this functional map indicates preferential connectivity between DGC and CA3 neurons and between CA3 and CA1 neurons, mimicking the neuronal circuitry of the intact hippocampus. We envision that patterned microislands, in combination with voltage imaging and methods to classify cell types, will be a powerful method for exploring neuronal function in both healthy and disease states. Additionally, because the entire neuronal network is sampled simultaneously, this strategy has the power to go further, revealing all functional connections between all cell types. Significance StatementIn vitro model systems provide unsurpassed control and access for exploring the molecular and cellular details of neurobiology. We developed a patterned microisland system for culturing rat hippocampal neurons that recapitulates the features of bulk hippocampal cultures, but with the added benefit of allowing access to high-speed imaging of entire neuronal ensembles using voltage imaging. By using far-red voltage-sensitive fluorophores, we map the functional connections across all cells in the neuronal ensemble, revealing that several important functional synapses present in the intact hippocampus are recapitulated in this microisland system. We envision the methods described here will be a powerful complement to ongoing research into basic neurobiological mechanisms and the search for therapies to treat diseases arising from their dysfunction.

neuroscience↗

bioRxiv2020

Integrated hiPSC-based liver and heart microphysiological systems predict unsafe drug-drug interaction

Microphysiological systems (MPSs) mimicking human organ function in vitro are an emerging alternative to conventional cell culture and animal models for drug development. Human induced pluripotent stem cells (hiPSCs) have the potential to capture the diversity of human genetics and provide an unlimited supply of cells. Combining hiPSCs with microfluidics technology in MPSs offers new perspectives for drug development. Here, the integration of a newly developed liver MPS with a cardiac MPS--both built with the same hiPSC line--to study drug-drug interaction (DDI) is reported. As a prominent example of clinically relevant DDI, the interaction of the arrhythmogenic gastroprokinetic cisapride with the fungicide ketoconazole was investigated. As seen in patients, metabolic conversion of cisapride to non-arrhythmogenic norcisapride in the liver MPS by the cytochrome P450 enzyme CYP3A4 was inhibited by ketoconazole, leading to arrhythmia in the cardiac MPS. These results establish functional integration of isogenic hiPSC-based liver and cardiac MPSs, which allows screening for DDI, and thus drug efficacy and toxicity, in the same genetic background.

bioengineering↗

bioRxiv2020

The npBAF to nBAF Chromatin Switch Regulates Cell Cycle Exit in the Developing Mammalian Cortex

Nervous system development is orchestrated by tightly-regulated progenitor cell divisions, followed by differentiation at precise but varying times across different regions. As progenitors exit the cell cycle, they initiate a subunit switch of the mSWI/SNF or npBAF complex to generate neuron-specific nBAF complexes. These chromatin regulatory complexes play dosage-sensitive roles in neural development and are frequently mutated in neurodevelopmental disorders. Here we manipulated the timing of BAF subunit exchange in the developing mouse brain and find that deletion of the npBAF subunit BAF53a blocks progenitor proliferation, leading to impaired neurogenesis. We show that npBAF complexes regulate cell cycle progression by antagonizing Polycomb complexes to promote chromatin accessibility at cell cycle and NPC identity genes. Replacement of the actin-related protein, Actl6a (BAF53a) by the neuron-specific actin-related protein, Actl6b (BAF53b), but not other regulators of proliferation, rescues progenitors by promoting neuronal differentiation. We propose that the function of the npBAF to nBAF chromatin switch is to control progenitor cell cycle exit and promote synchronous neural differentiation.

developmental biology↗

bioRxiv2019

Feasibility analysis of semiconductor voltage nanosensors for neuronal membrane potential sensing

In the last decade, optical imaging methods have significantly improved our understanding of the information processing principles in the brain. Although many promising tools have been designed, sensors of membrane potential are lagging behind the rest. Semiconductor nanoparticles are an attractive alternative to classical voltage indicators, such as voltage-sensitive dyes and proteins. Such nanoparticles exhibit high sensitivity to external electric fields via the quantum-confined Stark effect. Here we report the development of lipid-coated semiconductor voltage-sensitive nanorods (vsNRs) that self-insert into the neuronal membrane. We describe a workflow to detect and process the photoluminescent signal of vsNRs after wide-field time-lapse recordings. We also present data indicating that vsNRs are feasible for sensing membrane potential in neurons at a single-particle level. This shows the potential of vsNRs for detection of neuronal activity with unprecedentedly high spatial and temporal resolution.

bioengineering↗

bioRxiv2019

Inosine is an alternative carbon supply that supports effector T cell proliferation and anti-tumor function under glucose restriction

T cells undergo a characteristic metabolic rewiring that fulfills the dramatically increased bioenergetic, biosynthetic, and redox demands following antigen stimulation. A robust adaptive immune system requires effector T cells to respond and adapt to fluctuations in environmental nutrient levels imposed by infectious and inflammatory sites in different tissues. Inevitably, such responsiveness and adaptation reflect metabolic plasticity, allowing T cells to elicit immune functions by using a wide range of nutrient substrates. Here, we show that effector T cells utilize inosine, as an alternative substrate, to support cell growth and function in the absence of glucose. T cells metabolize inosine into hypoxanthine and phosphorylated ribose by purine nucleoside phosphorylase (PNP). Using Stable Isotope-Resolved Metabolomics (SIRM), we demonstrated that ribose moiety of inosine can enter into central metabolic pathways to provide ATP and biosynthetic precursors. Accordingly, the dependence of T cells on extracellular glucose for growth and effector functions can be relieved by inosine. On the other hand, cancer cells display diverse capacity to utilize inosine as a carbon resource. Moreover, the supplement of inosine enhances the anti-tumor efficacy of immune-checkpoint blockade or adoptive T cell transfer, reflecting the capability of inosine in relieving tumor-imposed metabolic restrictions on T cells in vivo.

immunology↗

bioRxiv2019

Symptom Severity in Youths with Attention Deficit Hyperactivity Disorder Associated with Normalizing Effects of Treatment on fMRI Response during a Stop Signal Task

Inhibitory control deficits represent one of many core cognitive deficits in Attention-Deficit/Hyperactivity Disorder (ADHD). Neuroimaging studies suggest that individuals with ADHD exhibit atypical engagement of neural systems during response inhibition, but the exact nature of this phenotype is obscured by mixed findings. We tested whether drug-free youths with ADHD (n=30, ages 7-14 years, 10 female) exhibited atypical neural correlates of response inhibition, as measured with a stop signal task and fMRI, compared to matched controls. We next investigated medication effects and whether there was a relationship between symptom severity and medication effects on the fMRI-evaluated signal. Finally, we tested for a significant difference between effects of monotherapy and combined pharmacological treatment. Patients showed significantly slower stop signal response time and lower percent inhibition, but no significant differences in the neural correlates of response inhibition relative to controls. However, patients showed significantly elevated signal in frontostriatal regions during responses. Prefrontal signal in patients was positively associated with reaction time variability in patients, and change (medicated - drug free) in the prefrontal signal was significantly associated with symptom scores, such that patients with elevated symptoms had greater BOLD signal reduction following treatment. Medication significantly improved go response time median and variability as well as stop signal reaction time, but there were no significant effects of medication or treatment type on BOLD signal. These findings challenge the notion of frontostriatal hypoactivation during response inhibition as a biomarker for ADHD and suggest that symptom severity may be associated with response to medication.

neuroscience↗

bioRxiv2019

Optical determination of absolute membrane potential

All cells maintain ionic gradients across their plasma membranes, producing transmembrane potentials (Vmem). Mounting evidence suggests a relationship between resting Vmem and the physiology of non-excitable cells with implications in diverse areas, including cancer, cellular differentiation, and body patterning. A lack of non-invasive methods to record absolute Vmem limits our understanding of this fundamental signal. To address this need, we developed a fluorescence lifetime-based approach (VF-FLIM) to visualize and optically quantify Vmem with single-cell resolution. Using VF-FLIM, we report Vmem distributions over thousands of cells, a 100-fold improvement relative to electrophysiological approaches. In human carcinoma cells, we visualize the voltage response to epidermal growth factor stimulation, stably recording a 10-15 mV hyperpolarization over minutes. Using pharmacological inhibitors, we identify the source of the hyperpolarization as the Ca2+-activated K+ channel Kca3.1. The ability to optically quantify absolute Vmem with cellular resolution will allow a re-examination of its roles as a cellular signal.

physiology↗