Optical Estimation of Membrane Potential Values Using Fluorescence Lifetime Imaging Microscopy and Hybrid Chemical-Genetic Voltage Indicators.

Introduction: Membrane potential (Vm), the voltage across a cell membrane, is an important biophysical phenomenon, central to the physiology of cells, tissues, and organisms. Voltage-sensitive fluorescent indicators are a powerful method for interrogating membrane potential in living systems, but most indicators are best suited for detecting changes in membrane potential rather than measuring values of the membrane potential. One promising approach is to use fluorescence lifetime imaging microscopy (FLIM) in combination of chemically synthesized dyes to estimate a value of membrane potential. However, a drawback is that chemically synthesized dyes show poor specificity of staining. Objectives: To address this problem, we applied a chemical-genetic voltage imaging approach to FLIM to enable optical estimation of membrane potential values from genetically defined cells. Results: In this report, we detail the characterization and evaluation of two of these systems in mammalian cells. We further validate the use of a FLIM-based chemical genetic voltage indicator in mammalian neurons. Conclusions: Finally, we discuss opportunities for future improvements to chemical-genetic FLIM-based voltage indicators.

Recording physiological history of cells with chemical labeling.

Recordings of the physiological history of cells provide insights into biological processes, yet obtaining such recordings is a challenge. To address this, we introduce a method to record transient cellular events for later analysis. We designed proteins that become labeled in the presence of both a specific cellular activity and a fluorescent substrate. The recording period is set by the presence of the substrate, whereas the cellular activity controls the degree of the labeling. The use of distinguishable substrates enabled the recording of successive periods of activity. We recorded protein-protein interactions, G protein-coupled receptor activation, and increases in intracellular calcium. Recordings of elevated calcium levels allowed selections of cells from heterogeneous populations for transcriptomic analysis and tracking of neuronal activities in flies and zebrafish.

Molecular Prosthetics for Long-Term Functional Imaging with Fluorescent Reporters.

Voltage-sensitive fluorescent reporters can reveal fast changes in the membrane potential in neurons and cardiomyocytes. However, in many cases, illumination in the presence of the fluorescent reporters results in disruptions to the action potential shape that limits the length of recording sessions. We show here that a molecular prosthetic approach, previously limited to fluorophores, rather than indicators, can be used to substantially prolong imaging in neurons and cardiomyocytes.

AlGaN/GaN asymmetric graded-index separate confinement heterostructures designed for electron-beam pumped UV lasers.

We present a study of undoped AlGaN/GaN separate confinement heterostructures designed to operate as electron beam pumped ultraviolet lasers. We discuss the effect of spontaneous and piezoelectric polarization on carrier diffusion, comparing the results of cathodoluminescence with electronic simulations of the band structure and Monte Carlo calculations of the electron trajectories. Carrier collection is significantly improved using an asymmetric graded-index separate confinement heterostructure (GRINSCH). The graded layers avoid potential barriers induced by polarization differences in the heterostructure and serve as strain transition buffers which reduce the mosaicity of the active region and the linewidth of spontaneous emission.

Solubility Limit of Ge Dopants in AlGaN: A Chemical and Microstructural Investigation Down to the Nanoscale.

Attaining low-resistivity AlxGa1-xN layers is one keystone to improve the efficiency of light-emitting devices in the ultraviolet spectral range. Here, we present a microstructural analysis of AlxGa1-xN/Ge samples with 0 ≤ x ≤ 1, and a nominal doping level in the range of 1020 cm-3, together with the measurement of Ge concentration and its spatial distribution down to the nanometer scale. AlxGa1-xN/Ge samples with x ≤ 0.2 do not present any sign of inhomogeneity. However, samples with x > 0.4 display µm-size Ge crystallites at the surface. Ge segregation is not restricted to the surface: Ge-rich regions with a size of tens of nanometers are observed inside the AlxGa1-xN/Ge layers, generally associated with Ga-rich regions around structural defects. With these local exceptions, the AlxGa1-xN/Ge matrix presents a homogeneous Ge composition which can be significantly lower than the nominal doping level. Precise measurements of Ge in the matrix provide a view of the solubility diagram of Ge in AlxGa1-xN as a function of the Al mole fraction. The solubility of Ge in AlN is extremely low. Between AlN and GaN, the solubility increases linearly with the Ga mole fraction in the ternary alloy, which suggests that the Ge incorporation takes place by substitution of Ga atoms only. The maximum percentage of Ga sites occupied by Ge saturates around 1%. The solubility issues and Ge segregation phenomena at different length scales likely play a role in the efficiency of Ge as an n-type AlGaN dopant, even at Al concentrations where Ge DX centers are not expected to manifest. Therefore, this information can have direct impact on the performance of Ge-doped AlGaN light-emitting diodes, particularly in the spectral range for disinfection (≈260 nm), which requires heavily doped alloys with a high Al mole fraction.

UV Emission from GaN Wires with m-Plane Core-Shell GaN/AlGaN Multiple Quantum Wells.

The present work reports high-quality nonpolar GaN/Al0.6Ga0.4N multiple quantum wells (MQWs) grown in core-shell geometry by metal-organic vapor-phase epitaxy on the m-plane sidewalls of c̅-oriented hexagonal GaN wires. Optical and structural studies reveal ultraviolet (UV) emission originating from the core-shell GaN/AlGaN MQWs. Tuning the m-plane GaN QW thickness from 4.3 to 0.7 nm leads to a shift of the emission from 347 to 292 nm, consistent with Schrödinger-Poisson calculations. The evolution of the luminescence with temperature displays signs of strong localization, especially for samples with thinner GaN QWs and no evidence of quantum-confined Stark effect, as expected for nonpolar m-plane surfaces. The internal quantum efficiency derived from the photoluminescence (PL) intensity ratio at low and room temperatures is maximum (∼7.3% measured at low power excitation) for 2.6 nm thick quantum wells, emitting at 325 nm, and shows a large drop for thicker QWs. An extensive study of the PL quenching with temperature is presented. Two nonradiative recombination paths are activated at different temperatures. The low-temperature path is found to be intrinsic to the heterostructure, whereas the process that dominates at high temperature depends on the QW thickness and is strongly enhanced for QWs larger than 2.6 nm, causing a rapid decrease in the internal quantum efficiency.

Role of Underlayer for Efficient Core-Shell InGaN QWs Grown on m-plane GaN Wire Sidewalls.

Different types of buffer layers such as InGaN underlayer (UL) and InGaN/GaN superlattices are now well-known to significantly improve the efficiency of c-plane InGaN/GaN-based light-emitting diodes (LEDs). The present work investigates the role of two different kinds of pregrowth layers (low In-content InGaN UL and GaN UL namely "GaN spacer") on the emission of the core-shell m-plane InGaN/GaN single quantum well (QW) grown around Si-doped c̅-GaN microwires obtained by silane-assisted metal organic vapor phase epitaxy. According to photo- and cathodoluminescence measurements performed at room temperature, an improved efficiency of light emission at 435 nm with internal quantum efficiency >15% has been achieved by adding a GaN spacer prior to the growth of QW. As revealed by scanning transmission electron microscopy, an ultrathin residual layer containing Si located at the wire sidewall surfaces favors the formation of high density of extended defects nucleated at the first InGaN QW. This contaminated residual incorporation is buried by the growth of the GaN spacer and avoids the structural defect formation, therefore explaining the improved optical efficiency. No further improvement is observed by adding the InGaN UL to the structure, which is confirmed by comparable values of the effective carrier lifetime estimated from time-resolved experiments. Contrary to the case of planar c-plane QW where the improved efficiency is attributed to a strong decrease of point defects, the addition of an InGaN UL seems to have no influence in the case of radial m-plane QW.

Spying on Neuronal Membrane Potential with Genetically Targetable Voltage Indicators.

Methods for optical measurement of voltage dynamics in living cells are attractive because they provide spatial resolution surpassing traditional electrode-based measurements and temporal resolution exceeding that of widely used Ca2+ imaging. Chemically synthesized voltage-sensitive dyes that use photoinduced electron transfer as a voltage-sensing trigger offer high voltage sensitivity and fast-response kinetics, but targeting chemical indicators to specific cells remains an outstanding challenge. Here, we present a new family of readily functionalizable, fluorescein-based voltage-sensitive fluorescent dyes (sarcosine-VoltageFluors) that can be covalently attached to a genetically encoded cell surface receptor to achieve voltage imaging from genetically defined neurons. We synthesized four new VoltageFluor derivatives that possess carboxylic acid functionality for simple conjugation to flexible tethers. The best of this new group of dyes was conjugated via a polyethylene glycol (PEG) linker to a small peptide (SpyTag, 13 amino acids) that directs binding and formation of a covalent bond with its binding partner, SpyCatcher (15 kDa). The new VoltageSpy dyes effectively label cells expressing cell-surface SpyCatcher, display good voltage sensitivity, and maintain fast-response kinetics. In cultured neurons, VoltageSpy dyes enable robust, single-trial optical detection of action potentials at neuronal soma with sensitivity exceeding genetically encoded voltage indicators. Importantly, genetic targeting of chemically synthesized dyes enables VoltageSpy to report on action potentials in axons and dendrites in single trials, tens to hundreds of micrometers away from the cell body. Genetic targeting of synthetic voltage indicators with VoltageSpy enables voltage imaging with low nanomolar dye concentration and offers a promising method for allying the speed and sensitivity of synthetic indicators with the enhanced cellular resolution of genetically encoded probes.

Fluorogenic Targeting of Voltage-Sensitive Dyes to Neurons.

We present a method to target voltage-sensitive fluorescent dyes to specified cells using an enzyme-catalyzed fluorogenic reaction on cell surfaces. The dye/enzyme hybrids are composed of a photoinduced electron transfer (PeT)-based fluorescent voltage indicator and a complementary enzyme expressed on the cell surface. Action of the exogenous enzyme on the dye results in fluorogenic activation of the dye, enabling fast voltage imaging in defined neurons with sensitivity surpassing those of purely genetically encoded approaches. We employ a bulky methylcyclopropylacetoxymethyl ether to diminish the fluorescence of a PeT-based voltage-sensitive dye, or VoltageFluor. The hydrolytically stable ether can be removed by the action of porcine liver esterase (PLE) to reveal the bright unmodified VoltageFluor. We established that the chemically modified VoltageFluor is a substrate for PLE in vitro and in live cells. When PLE is targeted to the external face of cell membranes, it controls the apparent staining of cells. The use of neuron-specific promoters can direct staining to mammalian neurons to provide clear detection of neuronal action potentials in single trials. All of the new VoltageFluors targeted by esterase expression (VF-EXs) report single spikes in cultured mammalian neurons. The best, VF-EX2, does so with a signal-to-noise ratio nearly double that of comparable genetically encoded voltage reporters. By targeting PLE to neurons, VF-EX2 can interrogate the neuromodulatory effects of serotonin in cultured hippocampal neurons. Taken together, our results show that a combination of synthetic chemistry and biochemistry enables bright and fast voltage imaging from genetically defined neurons in culture.

A Small-Molecule Photoactivatable Optical Sensor of Transmembrane Potential.

This paper discloses the design, synthesis, and imaging applications of the first member of a new class of SPOTs, small-molecule photoactivatable optical sensors of transmembrane potential. SPOT2.1.Cl features an established voltage-sensitive dye, VoltageFluor2.1.Cl--or VF--capped with a dimethoxy-o-nitrobenzyl (DMNB) caging group to effectively diminish fluorescence of the VF dye prior to uncaging. SPOT2.1.Cl localizes to cell membranes and displays weak fluorescence until photoactivated. Illumination generates the parent VF dye which then optically reports on changes in the membrane voltage. After photoactivation with spatially restricted light, SPOT2.1.Cl-loaded cells display bright, voltage-sensitive fluorescence associated with the plasma membrane, while neighboring cells remain dark. Activated SPOT reports on action potentials in single trials. SPOT can be activated in neuron cell bodies or uncaged in dendrites to enable structural tracing via "backfilling" of the dye to the soma, followed by functional imaging in the labeled cell. The combination of cellular specificity achieved through spatially defined patterns of illumination, coupled with the fast, sensitive, and noncapacitive voltage sensing characteristics of VF dyes makes SPOT2.1.Cl a useful tool for interrogating both structure and function of neuronal systems.

Enzymatic assay for GHB determination in forensic matrices.

Current procedures for the determination of gamma-hydroxybutyric acid (GHB) require time-consuming extraction and derivatization steps before chromatographic detection, making a high-throughput alternative desirable. Bühlmann Laboratories offers an enzymatic assay for the quantitative determination of GHB in urine and serum. We report the adaptation of this photometric assay to the Thermo Scientific MGC-240 analyzer and its use in the determination of GHB in forensic matrices including urine, whole blood and vitreous humour. Most matrices require only a brief centrifugation before analysis, while blood requires an additional protein precipitation step. A variety of cases (sexual assaults, impaired drivers and death investigations) have been analyzed alongside the gas chromatography-mass spectrometry (GC-MS) reference method. Correlation with the GC-MS has been found to be acceptable, with no false negatives and few false positives, although postmortem samples appear more prone to testing false positive than do antemortem samples. Simple sample preparation and high throughput allow for a significant reduction in analysis time relative to chromatographic methods. This assay is used as a screening method in our laboratory, with a quantitative GC-MS method serving for the confirmation of positive results. To our knowledge, this represents the first evaluation of an enzymatic assay for GHB in a forensic context.