Calcium Dye Research Articles

A single-compartment model of calcium dynamics in nerve terminals and dendrites.

This introduction describes a single-compartment model of calcium dynamics that has been applied to fluorescence measurements of intracellular free calcium concentration ([Ca(2+)]i) changes in neurons. The model describes intracellular calcium handling under simplified conditions, for which analytical expressions for the amplitude and the time constants of [Ca(2+)]i changes can be explicitly derived. In particular, it reveals the dependence of the measured [Ca(2+)]i changes on the calcium indicator concentration. Applied to experimental data from small cells or subcellular compartments, the model equations have been extremely useful for obtaining quantitative information about essential parameters of Ca(2+) influx, buffering, and clearance. We illustrate also several changes that occur when the basic assumptions do not hold (e.g., when calcium diffusion, dye saturation, or kinetic effects become significant). Finally, we discuss how the changes in calcium dynamics, which are explained by the model, have been exploited for measuring properties of calcium-driven reactions, such as those regulating short-term synaptic enhancement, vesicle recycling, and adaptation.

Measurement of shear stress-mediated intracellular calcium dynamics in human dermal lymphatic endothelial cells.

The shear stress applied to lymphatic endothelial cells (LEC) by lymph flow changes dramatically under normal conditions as well as in response to disease conditions and immune reactions. In general, LEC are known to regulate the contraction frequency and strength of lymphatic pumping in response to shear stress. Intracellular calcium concentration ([Ca2+]i) is an important factor that regulates lymphatic contraction characteristics. In this study, we measured changes in the [Ca2+]i under different shear stress levels and determined the source of this calcium signal. Briefly, human dermal LEC were cultured in custom-made microchannels for 3 days before loading with 2 µM fura-2 AM, a ratiometric calcium dye to measure [Ca2+]i. Step changes in shear stress resulted in a rapid increase in [Ca2+]i followed by a gradual return to the basal level and sometimes below the initial baseline (45.2 ± 2.2 nM). The [Ca2+]i reached a peak at 126.2 ± 5.6 nM for 10 dyn/cm2 stimulus, whereas the peak was only 71.8 ± 5.4 nM for 1 dyn/cm2 stimulus, indicating that the calcium signal depends on the magnitude of shear stress. Removal of the extracellular calcium from the buffer or pharmocological blockade of calcium release-activated calcium (CRAC) channels significantly reduced the peak [Ca2+]i, demonstrating a role of extracellular calcium entry. Inhibition of endoplasmic reticulum (ER) calcium pumps showed the importance of intracellular calcium stores in the initiation of this signal. In conclusion, we demonstrated that the shear-mediated calcium signal is dependent on the magnitude of the shear and involves ER store calcium release and extracellular calcium entry.

Intracellular calcium dynamics dependent on defined microtopographical features of titanium

Detailed insights into the complex cellular behavior at the biomaterial interface are crucial for the improvement of implant surfaces with respect to their acceptance and integration. The cells perceive microtopographical features and, in consequence, rearrange their adhesion structures like the actin cytoskeleton and adaptor proteins. But little is known about whether these altered cellular phenotypes have consequences for intracellular calcium signaling and its dynamics. To elucidate if an artificial, geometrical microtopography influences calcium ion (Ca2+) mobilization in osteoblasts, human MG-63 cells were stained with the calcium dye Fluo 3-acetoxymethyl ester and set on defined silicon–titanium (Ti) arrays with regular pillar structures (P5, 5 × 5 × 5 μm) and compared with planar Ti. To induce an immediate calcium signal, cells were stimulated with adenosine 5′-triphosphate (ATP). Interestingly, osteoblasts on micropillars expressing a shortened actin cytoskeleton were hampered in their calcium mobilization potential in signal height as well duration. Even the basal level of the intracellular Ca2+ concentration was reduced, which was accompanied by a disturbed fibronectin synthesis. The expression of the voltage-sensitive calcium channels Cav1.2, Cav1.3 (L-type) and Cav3.1, Cav3.2, Cav3.3 (T-type) as well as the signaling proteins phospho-AKT and phospho-GSK3α/β remained unaffected on pillars. The topography-dependent calcium dynamics observed here provide new insights into how topographical cues alter cell functions – via the intracellular Ca2+ signaling.

Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes.

At the frog neuromuscular junction, under physiological conditions, the direct measurement of calcium currents and of the concentration of intracellular calcium buffers—which determine the kinetics of calcium concentration and neurotransmitter release from the nerve terminal—has hitherto been technically impossible. With the aim of quantifying both Ca2+ currents and the intracellular calcium buffers, we measured fluorescence signals from nerve terminals loaded with the low-affinity calcium dye Magnesium Green or the high-affinity dye Oregon Green BAPTA-1, simultaneously with microelectrode recordings of nerve-action potentials and end-plate currents. The action-potential-induced fluorescence signals in the nerve terminals developed much more slowly than the postsynaptic response. To clarify the reasons for this observation and to define a spatiotemporal profile of intracellular calcium and of the concentration of mobile and fixed calcium buffers, mathematical modeling was employed. The best approximations of the experimental calcium transients for both calcium dyes were obtained when the calcium current had an amplitude of 1.6 ± 0.08 pA and a half-decay time of 1.2 ± 0.06 ms, and when the concentrations of mobile and fixed calcium buffers were 250 ± 13 μM and 8 ± 0.4 mM, respectively. High concentrations of endogenous buffers define the time course of calcium transients after an action potential in the axoplasm, and may modify synaptic plasticity.

Novel Genetically Encoded Ratiometric Calcium Indicators

Genetically encoded calcium indicators (GECIs) have recently undergone dramatic improvements in brightness, dynamic range, kinetics, and calcium sensitivity and now are capable of reporting single neuronal action potentials - comparing favorably to synthetic calcium dyes. Nevertheless, GECIs still lag behind synthetic calcium dyes in applications requiring quantitation of absolute intracellular calcium concentration. Here, we describe a novel approach to achieve dual-color ratiometric calcium measurements.When expressed individually, GCaMP and mCherry, respectively, exhibit calcium-dependent and -independent fluorescence intensities; and when present at the same level, their fluorescence intensity ratio should provide an estimate of absolute calcium concentration. Unfortunately, the two proteins do not always express equally in all locations in a cell. A simple tandem construct ensures equal levels everywhere of the two proteins, but non-radiative energy transfer from GCaMP to mCherry reduces the dynamic range of GCaMP signals and leads to apparent calcium-dependent fluorescence changes of mCherry. We introduced a rigid alpha helix, the ER/K helix, between the fluorophores, in order to prevent significant Forster resonance energy transfer (FRET). In parallel experiments, we demonstrated in vitro and in cellulo that the tandem GCaMP-ER/K-mCherry construct provides quantitative estimates of calcium concentration, without suffering reduction in dynamic range due to FRET activity.

Improving AM ester calcium dye loading efficiency.

Calcium imaging has unraveled the calcium-dependent mechanisms underlying neuronal function. Acetoxymethyl ester (AM) dyes are widely employed for calcium imaging. Pluronic F127 (PF127) as a surfactant and dimethyl sulfoxide (DMSO) as a solvent are used to dissolve the dyes, but concentrations vary between protocols. How these substances affect loading efficiency is not well characterized. We aimed to characterize dye loading in slice cultures. We determined minimum concentrations of surfactant, solvent and dye. The current study shows that the efficiency of AM dye loading depends on the initial stock concentration of PF127. Lowering the PF127 and DMSO concentrations can improve the loading efficiency. Both, pluronic and DMSO are required for successful dye loading. However, dissolving the dyes in lower concentrations of PF127 yielded better staining efficiency. Moreover, lowering the DMSO concentration to ∼0.25% improves the efficiency. The strategy allows standard two-photon or confocal microscope monitoring of neuronal activity. The labeled cells display spontaneous and evoked calcium transients, and repetitive measurements for up to 24h after loading indicate that the method is not deleterious to neuronal function. Dissolving the AM dyes in lower concentrations of PF127 combines the advantages of high loading efficiency, preserves cell viability and functional integrity, and allows repetitive measures over hours and days. Moreover, we found that the dye itself can be diluted to a final concentration of 1μM which reduces the experimental costs. The method is optimal for calcium imaging in slice cultures.

Wide-field in vivo neocortical calcium dye imaging using a convection-enhanced loading technique combined with simultaneous multiwavelength imaging of voltage-sensitive dyes and hemodynamic signals.

In vivo calcium imaging is an incredibly powerful technique that provides simultaneous information on fast neuronal events, such as action potentials and subthreshold synaptic activity, as well as slower events that occur in the glia and surrounding neuropil. Bulk-loading methods that involve multiple injections can be used for single-cell as well as wide-field imaging studies. However, multiple injections result in inhomogeneous loading as well as multiple sites of potential cortical injury. We used convection-enhanced delivery to create smooth, continuous loading of a large area of the cortical surface through a solitary injection site and demonstrated the efficacy of the technique using confocal microscopy imaging of single cells and physiological responses to single-trial events of spontaneous activity, somatosensory-evoked potentials, and epileptiform events. Combinations of calcium imaging with voltage-sensitive dye and intrinsic signal imaging demonstrate the utility of this technique in neurovascular coupling investigations. Convection-enhanced loading of calcium dyes may be a useful technique to advance the study of cortical processing when widespread loading of a wide-field imaging is required.

The effects of streptozocin‐induced type I diabetes on P2 receptors profile in the podocytes of the Dahl SS rat glomeruli (689.6)

Calcium flux in the podocytes is critical for their physiological function; excessive calcium in these cells can result in glomeruli damage, proteinuria and reduced GFR. P2 receptors are located throughout the kidney, and upon binding of ATP they activate calcium influx that triggers a plethora of intracellular processes. Our previous data have demonstrated that the major P2 receptor in the Sprague Dawley rat podocytes is P2Y1. Diabetic conditions have been shown to cause glomeruli injury and alter purinergic receptors profile in the kidney. The goal of the current study was to identify the P2 receptors responsible for calcium flux in the podocytes of the rats with type I diabetes. Type I diabetes was induced in the Dahl salt‐sensitive (SS) rats by an injection of streptozocin followed by an insulin implant, which results in the development of hyperfiltration, progressive proteinuria and renal damage typical for diabetic nephropathy. After 6 weeks of treatment, the glomeruli of the rats were isolated by differential sieving, loaded with Fluo‐4/FuraRed calcium dyes and analyzed with real‐time ratiometric fluorescent measurements. Pharmacological agonists and antagonists of the P2X and P2Y receptors were utilized to determine P2 receptors profile under diabetic conditions. This study will define the P2 receptors involved in the pathogenesis of the glomerular injury in diabetes, which could lead to a targeted treatment for renal complications associated with this disease.

Active Dendrites under Parental Supervision

Pyramidal cell dendrites are able to produce a variety of active calcium signals in brain slices. In this issue of Neuron, Grienberger etal. (2014) investigate dendritic function in the hippocampus of live mice.

Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes

The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sensor. These 'Twitch' sensors are based on the C-terminal domain of Opsanus troponin C. Their FRET responses were optimized by a large-scale functional screen in bacterial colonies, refined by a secondary screen in rat hippocampal neuron cultures. We tested the in vivo performance of the most sensitive variants in the brain and lymph nodes of mice. The sensitivity of the Twitch sensors matched that of synthetic calcium dyes and allowed visualization of tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes. Given their ratiometric readout, their brightness, large dynamic range and linear response properties, Twitch sensors represent versatile tools for neuroscience and immunology.

Activation of Intracellular Calcium by Multiple Wnt Ligands and Translocation of β-Catenin into the Nucleus: A CONVERGENT MODEL OF Wnt/Ca2+ AND Wnt/β-CATENIN PATHWAYS

Ca(2+) and β-catenin, a 92-kDa negatively charged transcription factor, transduce Wnt signaling via the non-canonical, Wnt/Ca(2+) and canonical, Wnt/β-catenin pathways independently. The nuclear envelope is a barrier to large protein entry, and this process is regulated by intracellular calcium [Ca(2+)]i and trans-nuclear potential. How β-catenin traverses the nuclear envelope is not well known. We hypothesized that Wnt/Ca(2+) and Wnt/β-catenin pathways act in a coordinated manner and that [Ca(2+)]i release facilitates β-catenin entry into the nucleus in mammalian cells. In a live assay using calcium dyes in PC3 prostate cancer cells, six Wnt peptides (3A, 4, 5A, 7A, 9B, and 10B) mobilized [Ca(2+)]i but Wnt11 did not. Based upon dwell time (range = 15-30 s) of the calcium waveform, these Wnts could be classified into three classes: short, 3A and 5A; long, 7A and 10B; and very long, 4 and 9B. Wnt-activated [Ca(2+)]i release was followed by an increase in intranuclear calcium and the depolarization of both the cell and nuclear membranes, determined by using FM4-64. In cells treated with Wnts 5A, 9B, and 10B, paradigm substrates for each Wnt class, increased [Ca(2+)]i was followed by β-catenin translocation into the nucleus in PC3, MCF7, and 253J, prostate, breast, and bladder cancer cell lines; both the increase in Wnt 5A, 9B, and 10B induced [Ca(2+)]i release and β-catenin translocation are suppressed by thapsigargin in PC3 cell line. We propose a convergent model of Wnt signaling network where Ca(2+) and β-catenin pathways may act in a coordinated, interdependent, rather than independent, manner.

Human inducible pluripotent stem cell-derived sensory neurons express multiple functional ion channels and GPCRs

Abstract Aims Aim of the study was to characterize functional ion channel and GPCR responses by using selective pharmacological tools and intracellular calcium imaging from human inducible pluripotent stem cell-derived sensory neurons. Methods Sensory neurons were generated from human keratinocytes that were reprogrammed to inducible pluripotent stem cells by using standard Yamanaka factors. Inducible pluripotent stem cells were differentiated into sensory neurons by using 2 differentiation protocols (small molecule and PA6 co-culture). Sensory neurons were loaded with intracellular calcium dye Fluo-4. Single-cell calcium imaging was performed with Photometrics Evolve EM-CCD camera at physiological temperature. Cells were perfused with a Ringer solution at 2–3 ml/min into which pharmacological compounds were dissolved. Data was analyzed with Till Photonics Offline Analysis program. Results Most of the results were obtained from PA6 differentiated neurons. 50 s application of 50 mM KCl solution was used as diagnostic tool to activate voltage-gated calcium channels and thereby evoke intracellular calcium elevation. Functional ASIC, NMDA, kainate and TRPA1 ion channels were present in a subset of sensory neurons. Majority of sensory neurons showed robust responses to purinergic stimulation with ATP and histaminergic stimulation with histamine, but not to subtype selective histamine H1, H2 or H4 stimulation suggesting the presence of H3 receptor subtype All cells responded strongly to protease-activated receptor stimulation with a low dose of trypsin. Interestingly, at single-cell level notable heterogeneity of ion channel and GPCR responses was observed. Conclusions Our results suggest that iPS-derived sensory neurons will be valuable in further pharmacological studies as well as sensory neuropathy disease modeling.

Monitoring Tectal Neuronal Activities and Motor Behavior in Zebrafish Larvae

To understand how visuomotor behaviors are controlled by the nervous system, it is necessary to monitor the activity of large populations of neurons with single-cell resolution over a large area of the brain in a relatively simple, behaving organism. The zebrafish larva, a small lower vertebrate with transparent skin, serves as an excellent model for this purpose. Immediately after the larva hatches, it needs to catch prey and avoid predators. This strong evolutionary pressure leads to the rapid development of functional sensory systems, particularly vision. By 5 d postfertilization (dpf), tectal cells show distinct visually evoked patterns of activation, and the larvae are able to perform a variety of visuomotor behaviors. During the early larval stage, zebrafish breathe mainly through the skin and can be restrained under the microscope using a drop of low-melting-point agarose, without the use of anesthetics. Moreover, the transparency of the skin, the small diameter of the neurons (4-5 µm), and the high-neuronal density enable the use of in vivo noninvasive imaging techniques to monitor neuronal activities of up to ∼500 cells within the central nervous system, still with single-cell resolution. This article describes a method for simultaneously monitoring spontaneous and visually evoked activities of large populations of neurons in the optic tectum of the zebrafish larva, using a synthetic calcium dye (Oregon Green BAPTA-1 AM) and a conventional confocal or two-photon scanning fluorescence microscope, together with a method for measuring the tail motor behavior of the head-immobilized zebrafish larva.

Monitoring of Single-Cell Responses in the Optic Tectum of Adult Zebrafish with Dextran-Coupled Calcium Dyes Delivered via Local Electroporation

The zebrafish (Danio rerio) has become one of the major animal models for in vivo examination of sensory and neuronal computation. Similar to Xenopus tadpoles neural activity in the optic tectum, the major region controlling visually guided behavior, can be examined in zebrafish larvae by optical imaging. Prerequisites of these approaches are usually the transparency of larvae up to a certain age and the use of two-photon microscopy. This principle of fluorescence excitation was necessary to suppress crosstalk between signals from individual neurons, which is a critical issue when using membrane-permeant dyes. This makes the equipment to study neuronal processing costly and limits the approach to the study of larvae. Thus there is lack of knowledge about the properties of neurons in the optic tectum of adult animals. We established a procedure to circumvent these problems, enabling in vivo calcium imaging in the optic tectum of adult zebrafish. Following local application of dextran-coupled dyes single-neuron activity of adult zebrafish can be monitored with conventional widefield microscopy, because dye labeling remains restricted to tens of neurons or less. Among the neurons characterized with our technique we found neurons that were selective for a certain pattern orientation as well as neurons that responded in a direction-selective way to visual motion. These findings are consistent with previous studies and indicate that the functional integrity of neuronal circuits in the optic tectum of adult zebrafish is preserved with our staining technique. Overall, our protocol for in vivo calcium imaging provides a useful approach to monitor visual responses of individual neurons in the optic tectum of adult zebrafish even when only widefield microscopy is available. This approach will help to obtain valuable insight into the principles of visual computation in adult vertebrates and thus complement previous work on developing visual circuits.

GABAergic signaling in the pulmonary neuroepithelial body microenvironment: functional imaging in GAD67-GFP mice

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates, but has also been reported in multiple cell types outside the CNS. A GABAergic system has been proposed in neuroepithelial bodies (NEBs) in monkey lungs. Pulmonary NEBs are known as complex intraepithelial sensory airway receptors and are part of the NEB microenvironment. Aim of the present study was to unravel a GABAergic signaling system in the NEB microenvironment in mouse lungs, enabling the use of genetically modified animals for future functional studies. Immunostaining of mouse lungs revealed that glutamic acid decarboxylase 65/67 (GAD65/67), a rate-limiting enzyme in the biosynthesis of GABA, and the vesicular GABA transporter (VGAT) were exclusively expressed in NEB cells. In GAD67-green fluorescent protein (GFP) knock-in mice, all pulmonary NEBs appeared to express GFP. For confocal live cell imaging, ex vivo vibratome lung slices of GAD67-GFP mice can be directly loaded with fluorescent functional probes, e.g. a red-fluorescent calcium dye, without the necessity of time-consuming prior live visualization of NEBs. RT-PCR of the NEB microenvironment obtained by laser microdissection revealed the presence of both GABAA and GABAB (R1 and R2) receptors, which was confirmed by immunostaining. In conclusion, the present study not only revealed the presence of a GABAergic signaling pathway, but also the very selective expression of GFP in pulmonary NEBs in a GAD67-GFP mouse model. Different proof of concept experiments have clearly shown that adoption of the GAD67-GFP mouse model will certainly boost future functional imaging and gene expression analysis of the mouse NEB microenvironment.

A Multiplexed Fluorescent Calcium and NFAT Reporter Gene Assay to Identify GPCR Agonists

Intracellular calcium response and resulting calcium signaling to an agonist-GPCR interaction are important for the measurement of compound activity in the GPCR drug development. The increase in cytosol calcium concentration can be measured by the fluorescent calcium indicator dye such as Fluo-4 in a quick assay (in 3-5 minutes) using the fluorescence imaging plate reader. The calcium signaling through the transcription factors such as NFAT that induces gene expression can be measured by the reporter gene assay that links to the expression of reporter enzyme such as the beta-lactamase that requires 5-hour incubation. We have evaluated a multiplexed assay that sequentially measures the calcium response to a GPCR agonist in a rapid fluorescent calcium dye assay, followed by a NFAT beta-lactamase assay, and compared them in the single assay format. We found that the agonist activity determined in the multiplexed assay were comparable with these determined in the single assay format and the Z’ factors were all >0.5. Five active compounds were identified that were active in both calcium dye assay and beta-lactamase assay. Therefore, our results demonstrated the utility of this multiplexed calcium assay for screening of GPCR compounds that can cross validate the primary hits and help to eliminate the false positive compounds.

The non‐neuronal cholinergic system: sources of vascular ACh

The localisation of muscarinic receptors in vascular endothelial cells and the consequences of activation of these receptors are extensively documented. However, it is not known if endogenous acetylcholine is available to activate receptors, and if so where it originates. As acetylcholine (ACh), its receptors and synthesising enzymes are widely expressed in extra‐neuronal cells and tissues (the non‐neuronal cholinergic system) including blood cells, we investigated a potential role for these cells as a source of endogenous ACh to stimulate the endothelium. Isolated rat mesenteric arteries were cut open and fixed en‐face. Endothelial cells were loaded with a calcium dye to measure levels of intracellular calcium using confocal microscopy. Endothelial cells exhibited transient but pronounced calcium increases when exposed to rat whole blood. The frequency of endothelial cell calcium events, average fluorescence and the number of active cells increased significantly above the spontaneous basal activity. ATP was shown not to be a component of this response, however, a trend in sensitivity to block with atropine (1μM) was observed. Taken together these data suggest that ACh may originate in sufficient quantities from blood cells to stimulate endothelial cell calcium increases. As such this may represent a significant auto or paracrine influence on vascular tone.Work funded by the British Heart Foundation.

Pharmacological characterization of the P2 receptors profile in the podocytes of the Sprague Dawley rat glomeruli

Renal P2 purinoreceptors are widely distributed throughout glomeruli, vasculature and tubular segments of the kidney. Extracellular ATP can mobilize calcium in the podocytes by activating these receptors and therefore contribute to glomeruli functions. The goal of this study was to identify specific P2 receptor subtypes in the podocytes of the rat glomeruli. The glomeruli of the Sprague Dawley rats were isolated by differential sieving, loaded with Fluo‐4/Fura Red calcium dyes and analysed with real‐time ratiometric fluorescent measurements under the confocal scope. Various specific chemical agonists and antagonists of the P2Y and P2X receptor subfamilies were utilized to determine responsible receptors under zero or 2 mM Ca2+ extracellular conditions. In contrast to previously published data, we have identified P2Y1 receptor as the main functional P2 receptor in the podocytes. Furthermore, we have assessed the contraction rates of the glomeruli in response to ATP and other P2Y1 receptors agonists therefore having shown the role of the P2 receptors, particularly, P2Y1, in the regulation of the glomerular volume dynamics. As a result of this study we identified P2Y1 as the main purinergic receptor localized in the podocytes, which might be involved in the pathogenesis of glomerular injury and in contraction and relaxation of glomeruli. Supported by the NIH (HL108880).

Functions and Imaging of Mast Cell and Neural Axis of the Gut

Close association between nerves and mast cells in the gut wall provides the microanatomic basis for functional interactions between these elements, supporting the hypothesis that a mast cell-nerve axis influences gut functions in health and disease. Advanced morphology and imaging techniques are now available to assess structural and functional relationships of the mast cell-nerve axis in human gut tissues. Morphologic techniques including co-labeling of mast cells and nerves serve to evaluate changes in their densities and anatomic proximity. Calcium (Ca(++)) and potentiometric dye imaging provide novel insights into functions such as mast cell-nerve signaling in the human gut tissues. Such imaging promises to reveal new ionic or molecular targets to normalize nerve sensitization induced by mast cell hyperactivity or mast cell sensitization by neurogenic inflammatory pathways. These targets include proteinase-activated receptor (PAR) 1 or histamine receptors. In patients, optical imaging in the gut in vivo has the potential to identify neural structures and inflammation in vivo. The latter has some risks and potential of sampling error with a single biopsy. Techniques that image nerve fibers in the retina without the need for contrast agents (optical coherence tomography and full-field optical coherence microscopy) may be applied to study submucous neural plexus. Moreover, the combination of submucosal dissection, use of a fluorescent marker, and endoscopic confocal microscopy provides detailed imaging of myenteric neurons and smooth muscle cells in the muscularis propria. Studies of motility and functional gastrointestinal disorders would be feasible without the need for full-thickness biopsy.

Imaging Large Cohorts of Single Ion Channels and Their Activity

As calcium is the most important signaling molecule in neurons and secretory cells, amongst many other cell types, it follows that an understanding of calcium channels and their regulation of exocytosis is of vital importance. Calcium imaging using calcium dyes such as Fluo3, or FRET-based dyes that have been used widely has provided invaluable information, which combined with modeling has estimated the subtypes of channels responsible for triggering the exocytotic machinery as well as inferences about the relative distances away from vesicle fusion sites these molecules adopt. Importantly, new super-resolution microscopy techniques, combined with novel Ca2+ indicators and imaginative imaging approaches can now define directly the nano-scale locations of very large cohorts of single channel molecules in relation to single vesicles. With combinations of these techniques the activity of individual channels can be visualized and quantified using novel Ca2+ indicators. Fluorescently labeled specific channel toxins can also be used to localize endogenous assembled channel tetramers. Fluorescence lifetime imaging microscopy and other single-photon-resolution spectroscopic approaches offer the possibility to quantify protein–protein interactions between populations of channels and the SNARE protein machinery for the first time. Together with simultaneous electrophysiology, this battery of quantitative imaging techniques has the potential to provide unprecedented detail describing the locations, dynamic behaviors, interactions, and conductance activities of many thousands of channel molecules and vesicles in living cells.