Real-Time Imaging of Electrical Signals with an Infrared FDA-Approved Dye

Clinical methods used to assess the electrical activity of excitable cells are often limited by their poor spatial resolution or their invasiveness. One promising solution to this problem is to optically measure membrane potential using a voltage-sensitive dye, but thus far, none of these dyes have been available for human use. Here we report that indocyanine green (ICG), an infrared fluorescent dye with FDA approval as an intravenously administered contrast agent, is voltage-sensitive. The fluorescence of ICG can follow action potentials in artificial neurons and cultured rat neurons and cardiomyocytes. ICG also visualized electrical activity induced in living explants of rat brain. In humans, ICG labels excitable cells and is routinely visualized transdermally with high spatial resolution. As an infrared voltage-sensitive dye with a low toxicity profile that can be readily imaged in deep tissues, ICG may have significant utility for clinical and basic research applications previously intractable for potentiometric dyes.Voltage-sensitive dyes provide a way to observe cellular electrical activity without the physical limitations imposed by electrodes. Although these dyes can monitor membrane potential with a resolution of a few microns from large populations of cells (1), there are three obstacles that prevent the use of these dyes in many research settings, including clinical research:

• 1.Most voltage-sensitive dyes use visible wavelengths of light that prevent imaging of tissues beneath the skin.
• 2.Many of these dyes produce significant toxicity or off-target effects (2).
• 3.Before this report, to our knowledge, no voltage-sensitive dyes have ever been available for administration in humans, which has limited their value in biomedically focused research.

Here, we show that indocyanine green (ICG), an FDA-approved fluorescent dye routinely used in many clinical tests, is voltage-sensitive. Our initial experimental system used Xenopus laevis oocytes. Changes in the membrane potential of the cell induced by two-electrode voltage-clamp resulted in robust, consistent changes in the fluorescence of ICG (Fig. 1, inset). All data in this work was obtained from single acquisitions with no averaging of multiple images. The voltage-dependent fluorescence changes were roughly linear with respect to membrane potential and had a magnitude of ∼1.9% of the baseline fluorescence per 100 mV of membrane potential change (Fig. 1). Additionally, ICG displayed a rapid response with a primary time constant of 4 ms (see Fig. S1 in the Supporting Material), suggesting that this dye could successfully monitor action potentials.Open in a separate windowFigure 1ICG-labeled oocytes showed that ICG’s fluorescence (blue points) is roughly linearly dependent (red line, fit to data) with voltage. (Inset) Oocyte membrane potential was held at −60 mV and then pulsed to potentials ranging from −120 mV (blue) to +120 mV (red). Ex: 780 nm, Em: 818–873 nm.To test this hypothesis, we transformed our oocytes into synthetic neurons, previously dubbed “excitocytes”, by coinjecting them with cRNA of voltage-gated sodium (Na_v) and potassium channel components (3). Under suitable current-clamp conditions, excitocytes fire trains of action potentials similar to those in naturally excitable cells. ICG’s fluorescence clearly recapitulated action potentials firing at speeds above 100 Hz (Fig. 2 A), faster than the physiological firing rates of most neurons (4).Open in a separate windowFigure 2ICG can monitor action potentials. (A) Oocytes coinjected with voltage-gated sodium and potassium channel cRNA fired action potentials (bottom, green) when held under current clamp. ICG fluorescence changes (top, blue) detected these action potentials at a rate of 107 Hz. Stimulus start (black arrow) and end (red arrow) are shown. (B and C) ICG fluorescence (blue, inverted) distinguished between healthy action potentials from wild-type sodium channels (B, green) and diseased action potentials from sodium channels with a myotonic substitution (C, green). Cells are stimulated for the entire time course of these panels. The delay between action potentials and the ICG signal is due to a low-pass filtering effect caused by the dye response time and the camera integration time. (D) In cells with myotonic sodium channels, a brief stimulus (top, black) was sufficient to elicit a train of action potentials (bottom, green) that only ceased upon significant hyperpolarization, as expected in a myotonia. ICG fluorescence (middle, blue) successfully followed each one of these action potentials.We extended the excitocyte technique from wild-type channels to evaluation of channelopathies and their effects on excitability to determine whether ICG could discriminate between normal and diseased action potentials based on shape. We compared excitocytes injected with wild-type Na_v channel cRNA to those injected with cRNA coding for a version of Na_v channel containing a point mutation, G1306E, which produces episodic myotonia (5). This disease is characterized by continued action potential firing in skeletal muscles after cessation of voluntary stimuli; the resulting prolonged muscle contractions are the hallmark of myotonia. Compared to the wild-type Na_v channel, the G1306E mutation causes a slowing of the fast inactivation of the Na_v channels, which in turn results in broadened action potentials (5). The electrical recordings and the ICG fluorescence response clearly distinguished the sharp action potentials produced by the healthy sodium channel (Fig. 2 B, and see Fig. S2) from the wider peaks produced by the myotonic sodium channel (Fig. 2 C, and see Fig. S2). Furthermore, a brief injection of current led to repetitive firing and hyperexcitability that persisted after the stimulus was stopped. ICG fluorescence clearly resolved every action potential of this myotonia-like behavior (Fig. 2 D). The successful recreation of disease-like action potentials validates the excitocyte system as a convenient method for investigating the electrophysiological effects of channelopathies.We next investigated whether ICG’s voltage sensitivity extended to excitable mammalian tissue. This validation was critical, inasmuch as other voltage-sensitive dyes have shown promise in invertebrate preparations but had much smaller signals in mammalian cells (6). We first measured ICG fluorescence from cultured rat dorsal root ganglion neurons. Under whole-cell current clamp, we observed neurons firing in the stereotypical fashion of the nociceptive C-type fiber, and these action potentials were clearly visible in the ICG fluorescence (Fig. 3 A, and see Fig. S3). We also examined syncytia of cultured cardiomyocytes from neonatal rats (7) to further validate ICG’s utility; these cells beat spontaneously and showed changes in ICG fluorescence indicative of changes in membrane potential (Fig. 3 B). Although we cannot formally exclude the possibility that the cardiomyocytes’ physical motion produced fluorescence changes, several observations suggested that these effects were minimal (see Fig. S4). Taken together, our results in frog and rat cells confirmed that ICG voltage sensitivity was broadly applicable across a range of tissues and not confined to a particular animal or cell lineage.Open in a separate windowFigure 3ICG follows electrical activity in living mammalian tissue. (A) Rat cultured dorsal root ganglion cells under current-clamp (black arrow, pulse start; red arrow, pulse end) fired action potentials (green), that ICG fluorescence tracked (blue, inverted, low-pass-filtered at 225 Hz; blue arrow, relative fluorescence change). (B) ICG fluorescence sensed spontaneous membrane potential changes in cardiomyocyte syncytia. (C) In rat brain slices, ICG responds differently to no stimulus (black) and stimuli of increasing intensity (magenta, cyan, green, and blue, increasing amplitude; scale bar shows relative fluorescence change). Weaker stimuli traces (e.g., magenta) show complete fluorescence recovery whereas larger stimuli (e.g., blue) do not fully recover within this time course; traces are vertically offset for clarity. (D) Tetrodotoxin (TTX) reduced the ICG response to a stimulus over 12 min (green, pre-TTX; cyan, magenta, and black, increasing time post-TTX; low-pass-filtered at 40 Hz; black arrow, stimulus).Finally, we tested whether ICG voltage sensitivity could be detected in a complex tissue. Rat hippocampal slice cultures comprise a well-described organotypic preparation in which the three-dimensional architecture, neuronal connections, and glial interactions are maintained (8,9). Using these rat brain explants, we found that brain excitation produced by field electrode stimulation was clearly accompanied by ICG fluorescence changes (see Fig. S5). Additionally, ICG discriminated between electrical responses caused by differing excitation intensities and durations (Fig. 3 C, and see Fig. S5). To confirm that the fluorescence changes originated from changes in excitable cell activity, we used the Na_v channel blocker tetrodotoxin (TTX). When applied to brain slices, electrical excitability was clearly inhibited (Fig. 3 D, and see Fig. S5) and partial recovery was observed upon subsequent TTX removal (see Fig. S5). These signals measuring brain slice activity were similar in shape and magnitude to those reported using other voltage-sensitive dyes (10,11). This demonstrates that ICG can report on electrical activity even in a physiological architecture with many nonexcitable cells.To our knowledge, this is the first report that a clinically approved fluorescent dye is voltage-sensitive. Our results demonstrate that indocyanine green can accurately detect action potentials at firing rates common in mammalian neurons, and that it is sensitive enough to distinguish between healthy and diseased action potentials in a model system. ICG can measure electrical activity in mammalian neurons, cardiomyocytes, and explanted brain tissue. This voltage sensitivity was observed with both monochromatic and broad-band illumination sources (data not shown), under labeling conditions that differed in solution composition, duration, and dye concentration (see Methods in the Supporting Material), and at temperatures ranging from 19°C to 30°C. ICG’s water solubility further extends its potential utility. This robustness suggests that ICG can be used to measure voltage in many environments and tissues.ICG has been FDA-approved for use in ophthalmic angiography, as well as in tests of cardiac output and hepatic function (12) and is additionally used off-label in a number of surgical applications (13). Interestingly, ICG has been shown to clearly label retinal ganglion cells in human patients (see Fig. S6) (14). This provides immediate motivation for biomedical investigations, because laboratory findings with ICG can potentially be translated to humans. Although many other voltage-sensitive dyes have been described, some with similar structures to ICG (15) and others with faster or larger signals (15,16), as of this writing none of these are FDA-approved. Additionally, ICG utilizes wavelengths further into the infrared spectrum than other available fast potentiometric dyes (17) and can thus be imaged in tissues up to 2 cm deep (18). This presents the possibility of optically imaging electrical activity deeper inside tissues than is feasible today. Although two-photon excitation with voltage-sensitive dyes can improve imaging depth, it remains intrinsically limited by the unaffected emission wavelength (19). Finally, ICG has been used in patients for more than 50 years and is known to have low toxicity (18,20). These properties suggest that ICG voltage sensitivity could extend the capabilities of modern electrophysiological techniques for disease diagnosis and monitoring in the clinic, and allow for the investigation of previously inaccessible experimental systems in basic research.     

用电压敏感染料光学记录膜电位

应用传统电生理方法如微电极和膜片钳技术,在记录较小的神经细胞和纤细的神经突起膜电位及同步记录神经细胞群的电活动等方面目前仍是一大难题。随着生理科学和神经生物学的发展,利用电压敏感染料光学记录膜电位技术已成为一种较为理想的新手段。本文对光学记录膜电位技术的发展史、染料特性和作用机制、光学成像及膜电位记录原理、目前的光学方法中某些不足及未来前景等做了较系统的介绍,并且简述了光学记录膜电位在电生理和神经生物学中的应用。     

Using voltage-sensitive dye recording to image the functional development of neuronal circuits in vertebrate embryos

Recent developments in the design of voltage-sensitive dyes and of recording apparatuses for detecting voltage-dependent changes in the optical properties of such dyes have established voltage-sensitive dye recording as an important technique for assessing the functional development of neuronal circuits in the brain and spinal cord. Here we discuss general technical issues regarding the recording of voltage-sensitive dye signals and describe studies that have utilized this approach to follow the development of sensory and sensorimotor circuits in the embryonic brain stem. Functional imaging through voltage-sensitive dye recording permits a noninvasive analysis of synaptic development and function at submillisecond temporal resolution in widely distributed circuits. These advantages are particularly valuable in assessing sensorimotor circuit development at early stages when neurons are small and synapses are fragile.     

Imaging Inhibitory Synaptic Potentials Using Voltage Sensitive Dyes

Studies of the spatio-temporal distribution of inhibitory postsynaptic potentials (IPSPs) in a neuron have been limited by the spatial information that can be obtained by electrode recordings. We describe a method that overcomes these limitations by imaging IPSPs with voltage-sensitive dyes. CA1 hippocampal pyramidal neurons from brain slices were loaded with the voltage-sensitive dye JPW-1114 from a somatic patch electrode in whole-cell configuration. After removal of the patch electrode, we found that neurons recover their physiological intracellular chloride concentration. Using an improved voltage-imaging technique, dendritic GABAergic IPSPs as small as 1 mV could be resolved optically from multiple sites with spatial averaging. We analyzed the sensitivity of the technique, in relation to its spatial resolution. We monitored the origin and the spread of IPSPs originating in different areas of the apical dendrite and reconstructed their spatial distribution. We achieved a clear discrimination of IPSPs from the dendrites and from the axon. This study indicates that voltage imaging is a uniquely suited approach for the investigation of several fundamental aspects of inhibitory synaptic transmission that require spatial information.     

Indocyanine green labeling for optical and photoacoustic imaging of mesenchymal stem cells after in vivo transplantation

The transplantation of mesenchymal stem cells (MSCs) holds great promise for the treatment of a plethora of human diseases, but new noninvasive procedures are needed to monitor the cell fate in vivo. Already largely used in medical diagnostics, the fluorescent dye indocyanine green (ICG) is an established dye to track limited numbers of cells by optical imaging (OI), but it can also be visualized by photoacoustic imaging (PAI), which provides a higher spatial resolution than pure near infrared fluorescence imaging (NIRF). Because of its successful use in clinical and preclinical examinations, we chose ICG as PAI cell labeling agent. Optimal incubation conditions were defined for an efficient and clinically translatable MSC labeling protocol, such that no cytotoxicity or alterations of the phenotypic profile were observed, and a consistent intracellular uptake of the molecule was achieved. Suspensions of ICG‐labeled cells were both optically and optoacoustically detected in vitro, revealing a certain variability in the photoacoustic spectra acquired by varying the excitation wavelength from 680 to 970 nm. Intramuscular engraftments of ICG‐labeled MSCs were clearly visualized by both PAI and NIRF over few days after transplantation in the hindlimb of healthy mice, suggesting that the proposed technique retains a considerable potential in the field of transplantation‐focused research and therapy. Stem cells were labeled with the Food and Drug Administration (FDA)‐approved fluorescent dye ICG, and detected by both PAI and OI, enabling to monitor the cell fate safely, in dual modality, and with good sensitivity and improved spatial resolution.      

Fast optical monitoring of microscopic excitation patterns in cardiac muscle.

Many vital processes depend on the generation, changes, and conduction of cellular transmembrane potentials. Optical monitoring systems are well suited to detect such cellular electrical activities in networks of excitable cells and also tissues simultaneously at multiple sites. Here, an exceptionally fast array system (16 x 16 photodiodes, up to 4,000,000 samples per second, 12-bit resolution) for imaging voltage-sensitive dye fluorescence, permitted real time measurements of excitation patterns at a microscopic size scale (256 pixels within an area of 1.8-8 mm2), in rat cardiac muscle in vitro. Results emphasize a recent hypothesis for cardiac impulse conduction, based on cardiac structural complexities, that is contradictory to all continuous cable theory models.     

Fast and aimed delivery of voltage-sensitive dyes to mammalian brain slices by biolistic techniques

Fast voltage-sensitive dyes (VSD) are widely used in modern neuroscience for optical recording of electrical potentials at many levels, from single cell compartment to brain areas, containing populations of many neural cells. The more lipophilic a VSD, the better signal-to-noise ratio of the optical signal, but there are no effective ways to deliver a water-insoluble dye into the membrane of live cell. Here we report a new protocol based on rapid biolistic delivery of VSDs, which is optimal for further recordings of optical signals from live neurons of rat brain slices. This protocol allows us to stain locally (150 mkm) neural somata of brain structures with a Golgi-like pattern, and a VSD propagates even to distant neurites of stained cells very quickly. This technique also can be used for rapid local delivery of any lipophilic and water-insoluble substances into live cells, further optical recording of neural activity, and analysis of potential propagation in a nerve cell.     

Genetically encoded fluorescent sensors of membrane potential

Imaging activity of neurons in intact brain tissue was conceived several decades ago and, after many years of development, voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome the drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a genetically encoded sensor could in principle be expressed specifically in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression. Here we critically review the current status of these developments.     

Imaging of respiratory-related population activity with single-cell resolution

The pre-B?tzinger complex (PBC) in the rostral ventrolateral medulla contains a kernel involved in respiratory rhythm generation. So far, its respiratory activity has been analyzed predominantly by electrophysiological approaches. Recent advances in fluorescence imaging now allow for the visualization of neuronal population activity in rhythmogenic networks. In the respiratory network, voltage-sensitive dyes have been used mainly, so far, but their low sensitivity prevents an analysis of activity patterns of single neurons during rhythmogenesis. We now have succeeded in using more sensitive Ca(2+) imaging to study respiratory neurons in rhythmically active brain stem slices of neonatal rats. For the visualization of neuronal activity, fluo-3 was suited best in terms of neuronal specificity, minimized background fluorescence, and response magnitude. The tissue penetration of fluo-3 was improved by hyperosmolar treatment (100 mM mannitol) during dye loading. Rhythmic population activity was imaged with single-cell resolution using a sensitive charge-coupled device camera and a x20 objective, and it was correlated with extracellularly recorded mass activity of the contralateral PBC. Correlated optical neuronal activity was obvious online in 29% of slices. Rhythmic neurons located deeper became detectable during offline image processing. Based on their activity patterns, 74% of rhythmic neurons were classified as inspiratory and 26% as expiratory neurons. Our approach is well suited to visualize and correlate the activity of several single cells with respiratory network activity. We demonstrate that neuronal synchronization and possibly even network configurations can be analyzed in a noninvasive approach with single-cell resolution and at frame rates currently not reached by most scanning-based imaging techniques.     

Optical indications of electrical activity and excitation-contraction coupling in the early embryonic heart

Complete understanding of the ontogenesis and early development of electrical activity and its related contraction has been hampered by our inability to apply conventional electrophysiological techniques to the early embryonic heart. Direct intracellular measurement of electrical events in the early embryonic heart is impossible because the cells are too small and frail to be impaled with microelectrodes. Optical signals from voltage-sensitive dyes have provided a new and powerful tool for monitoring changes in membrane potential in a wide variety of living preparations. With this technique it is possible to make optical recordings from cells which are inaccessible to microelectrodes. An additional advantage of the optical method for recording membrane potential activity is that electrical activity can be monitored simultaneously from many sites in a preparation. Thus, applying a multiple-site optical recording method with a 100- or 144-element photodiode array and voltage-sensitive dyes, we have been able to monitor for the first time spontaneous electrical activity in pre-fused cardiac primordia in early chick embryos at the 6- and early 7-somite stages of development; we have been able to determine that the time of initiation of the heartbeat is the middle period of the 9-somite stage. In the rat embryonic heart, the onset of spontaneous electrical activity and contraction occurs at the 3-somite stage. This article describes ionic properties of the spontaneous action potential and genesis of excitation-contraction coupling in the early embryonic chick and rat hearts. In addition, an improved view of the ontogenetic sequence of spontaneous electrical activity and its implications for excitation-contraction coupling in the early embryonic heart are proposed and discussed.     

Imaging Brain Activity With Voltage- and Calcium-Sensitive Dyes

This paper presents three examples of imaging brain activity with voltage- or calcium-sensitive dyes and then discusses the methodological aspects of the measurements that are needed to achieve an optimal signal-to-noise ratio.Internally injected voltage-sensitive dye can be used to monitor membrane potential in the dendrites of invertebrate and vertebrate neurons in in vitro preparations.Both invertebrate and vertebrate ganglia can be bathed in voltage-sensitive dyes to stain all of the cell bodies in the preparation. These dyes can then be used to follow the spike activity of many neurons simultaneously while the preparations are generating behaviors.Calcium-sensitive dyes that are internalized into olfactory receptor neurons in the nose will, after several days, be transported to the nerve terminals of these cells in the olfactory bulb. There they can be used to measure the input from the nose to the bulb.Three kinds of noise are discussed. a. Shot noise from the random emission of photons from the preparation. b. Vibrational noise from external sources. c. Noise that occurs in the absence of light, the dark noise.Three different parts of the light measuring apparatus are discussed: the light sources, the optics, and the cameras.The major effort presently underway to improve the usefulness of optical recordings of brain activity are to find methods for staining individual cell types in the brain. Most of these efforts center around fluorescent protein sensors of activity.     

Quantitative imaging of the heterogeneity of membrane activation of mammalian neurons and glial cells

By using quantitative imaging with an ultra-high sensitivity, it was possible to observe the simultaneous action of multiple patches unevenly distributed over the membranes of neurons and glial cells in culture. We used a voltage-sensitive probe to stain vitally the cells. The instrumentation consisted of a liquid-nitrogen cooled matrix of 222,530 photodetectors with a spatial resolution of 0.25 microns 2, a photodynamic range of 10(5), a detection level of a few tens of photons and a maximum time resolution of 500 microseconds. Electrical and pharmacological stimulations were applied to produce the activation of the cells which was accompanied by large variations of the level of fluorescence, giving a precise spatial localization of active domains over the soma-neuritic membranes. These images of fluorescent signals are interpreted as corresponding to the plasmalemmal localization of voltage-dependent channels. This finding, which had not been previously observed with voltage-sensitive probes in fluorescent dye imaging indicates the possibility of measuring the activity of independently functioning domains in single neurons.     

Imaging activity of neuronal populations with new long-wavelength voltage-sensitive dyes

We have assessed the utility of five new long-wavelength fluorescent voltage-sensitive dyes (VSD) for imaging the activity of populations of neurons in mouse brain slices. Although all the five were capable of detecting activity resulting from activation of the Schaffer collateral-CA1 pyramidal cell synapse, they differed significantly in their properties, most notably in the signal-to-noise ratio of the changes in dye fluorescence associated with neuronal activity. Two of these dyes, Di-2-ANBDQPQ and Di-1-APEFEQPQ, should prove particularly useful for imaging activity in brain tissue and for combining VSD imaging with the control of neuronal activity via light-activated proteins such as channelrhodopsin-2 and halorhodopsin.     

VSDI: a new era in functional imaging of cortical dynamics

During the last few decades, neuroscientists have benefited from the emergence of many powerful functional imaging techniques that cover broad spatial and temporal scales. We can now image single molecules controlling cell differentiation, growth and death; single cells and their neurites processing electrical inputs and sending outputs; neuronal circuits performing neural computations in vitro; and the intact brain. At present, imaging based on voltage-sensitive dyes (VSDI) offers the highest spatial and temporal resolution for imaging neocortical functions in the living brain, and has paved the way for a new era in the functional imaging of cortical dynamics. It has facilitated the exploration of fundamental mechanisms that underlie neocortical development, function and plasticity at the fundamental level of the cortical column.     

Dye-coupling visualizes networks of large-field motion-sensitive neurons in the fly

In the fly, visually guided course control is accomplished by a set of 60 large-field motion-sensitive neurons in each brain hemisphere. These neurons have been shown to receive retinotopic motion information from local motion detectors on their dendrites. In addition, recent experiments revealed extensive coupling between the large-field neurons through electrical synapses. These two processes together give rise to their broad and elaborate receptive fields significantly surpassing the extent of their dendritic fields. Here, we demonstrate that the electrical connections between different large-field neurons can be visualized using Neurobiotin dye injection into a single one of them. When combined with a fluorescent dye which does not cross electrical synapses, the injected cell can be identified unambiguously. The Neurobiotin staining corroborates the electrical coupling postulated amongst the cells of the vertical system (VS-cells) and between cells of the horizontal system (HS-cells and CH-cells). In addition, connections between some cells are revealed that have so far not been considered as electrically coupled.     

Voltage-sensitive dye recording of action potentials and synaptic potentials from sympathetic microcultures.

Given the appropriate multicell electrophysiological techniques, small networks of cultured neurons (microcultures) are well suited to long-term studies of synaptic plasticity. To this end, we have developed an apparatus for optical recording from cultured vertebrate neurons using voltage-sensitive fluorescent dyes (Chien, C.-B., and J. Pine. 1991. J. Neurosci. Methods. 38:93-105). We evaluate here the usefulness of this technique for recording action potentials and synaptic potentials in microcultures of neurons from the rat superior cervical ganglion (SCG). After extensive dye screening and optimization of conditions, we chose the styryl dye RH423, which gave fast linear fluorescence changes of approximately 1%/100 mV for typical recordings. The root mean square noise of the apparatus (limited by shot noise) was typically 0.03%, equivalent to 3 mV of membrane potential. Illumination for at least 100 flashes of 100 ms each caused no noticeable photodynamic damage. Our results show that voltage-sensitive dyes can be used to record from microcultures of vertebrate neurons with high sensitivity. Dye signals were detected from both cell bodies and neurites. Signals from presumptive dendrites showed hyperpolarizations and action potentials simultaneous with those in the cell body, while those from presumptive axons showed delayed propagating action potentials. Subthreshold synaptic potentials in the cell body were occasionally detectable optically; however, they were usually masked by signals from axons passing through the same pixel. This is due to the complex anatomy of SCG microcultures, which have many crisscrossing neurites that often pass over cell bodies. Given a simpler microculture system with fewer neurites, it should be possible to use dye recording to routinely measure subthreshold synaptic strengths.     

Nasal chemosensory-stimulation evoked activity patterns in the rat trigeminal ganglion visualized by in vivo voltage-sensitive dye imaging

Mammalian nasal chemosensation is predominantly mediated by two independent neuronal pathways, the olfactory and the trigeminal system. Within the early olfactory system, spatiotemporal responses of the olfactory bulb to various odorants have been mapped in great detail. In contrast, far less is known about the representation of volatile chemical stimuli at an early stage in the trigeminal system, the trigeminal ganglion (TG), which contains neurons directly projecting to the nasal cavity. We have established an in vivo preparation that allows high-resolution imaging of neuronal population activity from a large region of the rat TG using voltage-sensitive dyes (VSDs). Application of different chemical stimuli to the nasal cavity elicited distinct, stimulus-category specific, spatiotemporal activation patterns that comprised activated as well as suppressed areas. Thus, our results provide the first direct insights into the spatial representation of nasal chemosensory information within the trigeminal ganglion imaged at high temporal resolution.     

Nano to Micro — Fluorescence Measurements of Electric Fields in Molecules and Genetically Specified Neurons

Our central nervous system is based on the generation and propagation of electrical signals along the neuronal pathways. These variations of the membrane potential are arranged by the concerted action of ion channels in the neuronal membrane. Therefore, the exact measurement of the electric field in the central nervous system is the focus of intensive investigation. While electrophysiological methods provide exact measurements on the single-cell or single-molecule level with high temporal resolution, they are limited in their spatial resolution ranging from a few single cells to a single molecule. To thoroughly understand how the voltage-dependent ion channels sense the membrane potential and are precisely gated by it, the electric field within the protein has to be investigated. Likewise, the propagation of electrical impulses in a network of neurons involves a large number of cells, which have to be monitored simultaneously. For these endeavors, optical methods have proven to be useful due to their scalability, temporal and spatial resolution. Here, we will summarize the properties of the optical probes that we used to determine the electrical field strength within voltage-sensitive ion channels and discuss the hybrid approach to detect membrane potential changes in genetically specified neurons in terms of design, limitations and future developments.     

Thermoacoustic molecular imaging of small animals

We have designed, constructed, and tested a thermoacoustic computed tomography (TCT) scanner for imaging optical absorption in small animals in three dimensions. The device utilizes pulsed laser irradiation (680-1064 nm) and a unique, 128-element transducer array. We quantified the isotropic spatial resolution of this scanner to be 0.35 mm. We describe a dual-wavelength subtraction technique for isolating optical dyes with TCT. Phantom experiments demonstrate that we can detect 5 fmol of a near-infrared dye (indocyanine green, ICG) in a 1-microL volume using dual-wavelength subtraction. Initial TCT imaging in phantoms and in two sacrificed mice suggests that three-dimensional, optical absorption patterns in small animals can be detected with an order of magnitude better spatial resolution and an order of magnitude better low-contrast detectability in small animals when compared to fluorescence imaging or diffusion optical tomography.