Potentiometric Probes Research Articles

High-throughput methods for cardiac cellular electrophysiology studies: the road to personalized medicine.

Personalized medicine refers to the tailored application of medical treatment at an individual level, considering the specific genotype or phenotype of each patient for targeted therapy. In the context of cardiovascular diseases, implementing personalized medicine is challenging due to the high costs involved and the slow pace of identifying the pathogenicity of genetic variants, deciphering molecular mechanisms of disease, and testing treatment approaches. Scalable cellular models such as human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) serve as useful in vitro tools that reflect individual patient genetics and retain clinical phenotypes. High-throughput functional assessment of these constructs is necessary to rapidly assess cardiac pathogenicity and test new therapeutics if personalized medicine is to become a reality. High-throughput photometry recordings of single cells coupled with potentiometric probes offer cost-effective alternatives to traditional patch-clamp assessments of cardiomyocyte action potential characteristics. Importantly, automated patch-clamp (APC) is rapidly emerging in the pharmaceutical industry and academia as a powerful method to assess individual membrane-bound ionic currents and ion channel biophysics over multiple cells in parallel. Now amenable to primary cell and hiPSC-CM measurement, APC represents an exciting leap forward in the characterization of a multitude of molecular mechanisms that underlie clinical cardiac phenotypes. This review provides a summary of state-of-the-art high-throughput electrophysiological techniques to assess cardiac electrophysiology and an overview of recent works that successfully integrate these methods into basic science research that could potentially facilitate future implementation of personalized medicine at a clinical level.

Self-powered optical ion sensor array based on potentiometric probes coupled to electronic paper

This work describes a direct optical readout of an ion-selective potentiometric sensing array without an external power supply. Electronic paper (e-paper) was chosen as the signal transducer owing to its fast response on the order of seconds and its wide dynamic range of about 1.5 V. The capacitive character of the display allows one to observe the optical response at zero current, which is a desired condition for the potentiometric measurement. An applied potential of 1 V gives a transient charge flow of about 11 μC for the e-paper to exhibit a maximal intensity change. The cathodes of three different pixels of the e-paper were connected to three different ISEs responsive to Na+, K+, and Ca2+, while their common anode was connected to the shared reference electrode (RE). In this manner, each pixel is mapped to the behavior of one sensor, making it possible to detect multiple ions with an e-pixel array. The sensor configuration gave quantitative information on Na+, K+, and Ca2+ from 10−5 M to 10−1 M by analyzing the RGB information of the pixels. The e-paper exhibited a stable response within half a minute, much faster than previously established electrochromic material transducers such as Prussian Blue (PB). With this approach we successfully carried out multi-ion concentration sensing without external power, which is potentially attractive for applications in environmental monitoring, clinical assays and wearable sensing devices.

Current Practice in Using Voltage Imaging to Record Fast Neuronal Activity: Successful Examples from Invertebrate to Mammalian Studies.

The optical imaging of neuronal activity with potentiometric probes has been credited with being able to address key questions in neuroscience via the simultaneous recording of many neurons. This technique, which was pioneered 50 years ago, has allowed researchers to study the dynamics of neural activity, from tiny subthreshold synaptic events in the axon and dendrites at the subcellular level to the fluctuation of field potentials and how they spread across large areas of the brain. Initially, synthetic voltage-sensitive dyes (VSDs) were applied directly to brain tissue via staining, but recent advances in transgenic methods now allow the expression of genetically encoded voltage indicators (GEVIs), specifically in selected neuron types. However, voltage imaging is technically difficult and limited by several methodological constraints that determine its applicability in a given type of experiment. The prevalence of this method is far from being comparable to patch clamp voltage recording or similar routine methods in neuroscience research. There are more than twice as many studies on VSDs as there are on GEVIs. As can be seen from the majority of the papers, most of them are either methodological ones or reviews. However, potentiometric imaging is able to address key questions in neuroscience by recording most or many neurons simultaneously, thus providing unique information that cannot be obtained via other methods. Different types of optical voltage indicators have their advantages and limitations, which we focus on in detail. Here, we summarize the experience of the scientific community in the application of voltage imaging and try to evaluate the contribution of this method to neuroscience research.

Near infrared voltage sensitive dyes based on chromene electron donors.

Excellent hemicyanine voltage sensitive dyes (VSDs) are available for optical electrophysiology over a wide spectral range out to the far red. Near infrared fluorescent VSDs would be advantageous because they allow deeper light penetration into tissue because of decreased light scattering and also because they can be used in combination with other fluorescent indicators or optogenetic actuators. The usual approach to constructing long wavelength chromophores is to lengthen the π-system; however, this is generally accompanied by a significant decrease in fluorescence quantum efficiency and can also decrease aqueous solubility, making it difficult to deliver the probes to the target tissue. The VSDs developed in our lab are generally composed of an amino-naphthyl electron donor moiety linked to a pyridinium acceptor. Here we show that replacing the naphthalene with chromene induces a 60-80 nm red-shift in the absorption and emission spectra while maintaining the structure at the same size and fluorescence quantum yield at the same level. The voltage sensitivity of these chromene-based VSDs is also comparable to our older VSDs and they display opposite polarity sensitivity when excited at the short and long wavelength edges of the absorbance spectra; thus they can be used in dual wavelength ratio measurements to eliminate artifacts from motion and bleaching. We have tested one of these VSDs in voltage imaging of a whole murine heart at 730 nm, by far the longest wavelength reported in voltage imaging. It resolves action potentials in single sweep scans with 10.1% ΔF/F per action potential. Chromene-based electron donors are likely to find wide applications in generating long wavelength hemicyanines or cyanines. (Supported by NIH grant MH116830. Conflict of Interest statement: LML, CDA, and PY are founders and owners of Potentiometric Probes LLC.)

Reference Systems for Lithium Sensors in Molten Alloys

Tritium is one of the required elements in future fusion reactors. This hydrogen isotope has to be continuously produced to supply the fusion reactor. Tritium can be obtained from the fusion reaction of lithium 6. Because of that, future fusion reactors are designed to have a tritium breeding module surrounding the reactor. One of the breeding blanket designs that has been proposed is based on lead-lithium eutectic melt [1]. Lithium monitoring in the breeding blanket is of great importance for the performance of the reactor. A deviation from the eutectic composition can cause a substantial change in tritium production.To control the lithium dosage needed and keep the adequate composition in the melt, online lithium measurements will be required. For this purpose, lithium probes must be designed to withstand the high temperatures of the breeding modules (~400 ºC) and the chemical reactivity of lithium. The constructed sensors need to be tested at different lithium concentrations to determine their viability and range of application.Sensors based on solid-state electrolytes have several advantages: stable compounds which can withstand the harsh chemical environment of the melts, their ionic conductivity tends to increase with the temperature [2] and the output signal (cell potential or electrochemical current) is easy to measure. Lithium conducting electrolytes for molten metals are under development at the Electrochemical Methods Laboratory at Institut Quimic de Sarria (IQS), Barcelona. Its qualification and performance are being tested.In this work, Li6BaLa2Ta2O12 [3], a lithium-ion conducting solid-state electrolyte, has been used to develop potentiometric probes capable of measuring Li in molten Pb-Li alloys. As working electrode, Pb-Li alloys whose concentrations were between 3at% and 17at% Li were used. As reference electrode, Sn-Li and Pb-Li alloys were tested and compared. In these alloys, Li at% composition was kept constant during the whole experiment. Potentiometric measurements were performed at three different temperatures: 400 ºC, 500 ºC, and 600 ºC. Experimental data were in good agreement with theoretical calculations.[1] F. De Schutter, J. Dekeyser, J. Luyten, H. Tas, Electrochemical sensors for oxygen and lithium detection in Pb17Li, Fusion Eng. Des. 14 (1991) 241–248. https://doi.org/10.1016/0920-3796(91)90008-E.[2] V. Thangadurai, S. Narayanan, D. Pinzaru, Garnet-type solid-state fast Li ion conductors for Li batteries: critical review, Chem. Soc. Rgev. 43 (2014) 4714. https://doi.org/10.1039/c4cs00020j.[3] M. Nel-lo, S. Colominas, J. Abellà, Lithium conducting ceramics for future electrochemical sensors in molten metals, Fusion Eng. Des. (2019). https://doi.org/10.1016/j.fusengdes.2019.02.043.

Use of Amperometric and Potentiometric Probes in Scanning Electrochemical Microscopy for the Spatially-Resolved Monitoring of Severe Localized Corrosion Sites on Aluminum Alloy 2098-T351.

Amperometric and potentiometric probes were employed for the detection and characterization of reactive sites on the 2098-T351 Al-alloy (AA2098-T351) using scanning electrochemical microscopy (SECM). Firstly, the probe of concept was performed on a model Mg-Al galvanic pair system using SECM in the amperometric and potentiometric operation modes, in order to address the responsiveness of the probes for the characterization of this galvanic pair system. Next, these sensing probes were employed to characterize the 2098-T351 alloy surface immersed in a saline aqueous solution at ambient temperature. The distribution of reactive sites and the local pH changes associated with severe localized corrosion (SLC) on the alloy surface were imaged and subsequently studied. Higher hydrogen evolution, lower oxygen depletion and acidification occurred at the SLC sites developed on the 2098-T351 Al-alloy.

Permeability-Controllable Potentiometric Fluorescent Probes Enable Visually Discriminating Near-Zero and Normal Situations of Cell Membrane Potential.

The permeability-controllable potentiometric fluorescent probes that can visually discriminate near-zero and normal situations of cell membrane potential were reported for the first time. Different from traditional potentiometric probes that utilize fluorescence intensity to reflect membrane potential, CQ12 and CP12 have different localizations under the two situations of cell membrane potential. Thus, the two situations can be point-to-point indicated by two fluorescent images with an obvious difference, avoiding complex operations and calibration of conventional methods.

Feasibility of spectral pH measurement during the low-pH virus inactivation step of continuous therapeutic antibody production.

Acidic virus inactivation is commonly used during production of biotherapeutic products to provide virus safety in case of undetected virus contamination. Accurate pH measurement is required to ensure the product pH reaches a virus-inactivating level (typically 3.5-3.7), and a level post-inactivation that is appropriate for later purification steps (typically 5.5-7.5). During batch low-pH inactivation in discrete tanks, potentiometric glass probes are appropriate for measuring pH. During continuous inactivation for 2-3 weeks in an enclosed product stream, probe calibration drift and lag may lead to poor accuracy, and operational difficulties when compensating for drift. Monitoring the spectral response of compounds (indicators) in the product stream whose spectra are pH-sensitive offers a possible alternative way to measure pH without these drawbacks. Such indicators can already exist in the stream (intrinsic) or can be added (extrinsic). Herein are reported studies evaluating the feasibility of both.Promising ultraviolet screening results with the two extrinsics studied, thiamine and ascorbic acid, led to the addition of both to product stream samples titrated to different potentiometric pH values in the 3.3-4.5 range (a representative range encountered during continuous inactivation), and attempts to model pH using sample ultraviolet spectra. One model, based on variability in six spectral attributes, was able to predict pH of an independent sample set within ±0.07 units at the 95% confidence level. Since a typical inactivating pH tolerance is ±0.1 units, the results show that extrinsic indicators potentially can measure inactivation pH with sufficient accuracy. Suggested future steps and an alternative approach are presented.

Ultrasensitive Seawater pH Measurement by Capacitive Readout of Potentiometric Sensors.

Potentiometric pH probes remain the gold standard for the detection of pH but are not sufficiently sensitive to reliably detect ocean acidification at adequate frequency. Here, potentiometric probes are made dramatically more sensitive by placing a capacitive electronic component in series to the pH probe while imposing a constant potential over the measurement circuit. Each sample change now triggers a capacitive current transient that is easily identified between the two equilibrium states, and is integrated to reveal the accumulated charge. This affords dramatically higher precision than with traditional potentiometric probes. pH changes down to 0.001 pH units are easily distinguished in buffer and seawater samples, at a precision (standard deviation) of 28 μpH and 67 μpH, respectively, orders of magnitude better than what is possible with potentiometric pH probes.

Direct Potentiometric Sensing of Anion Concentration (Not Activity).

Potentiometric probes used in direct potentiometry are attractive sensing tools. They give information on ion activities, which is often uniquely useful. If, instead, concentrations are desired as sensor output, the ionic strength of the sample must be precisely known, which is often not possible. Here, for the first time, direct potentiometry can be made to report concentrations, rather than activities. It is demonstrated for the detection of monovalent anionic species by using a self-referencing Ag/AgI pulstrode as the reference element instead of a traditional reference electrode. This reference pulstrode releases a discrete quantity of iodide ions from the electrode and the resulting reference potential varies with the activity coefficient of iodide. The effects of activity coefficient on the indicator and reference electrode are therefore compensated and the observed cell potential may now be described in a Nernstian manner against anion concentration, rather than activity. Theoretical simulations and experimental results support the validity of this approach. For most monovalent anions of practical relevance, the potential difference between this approach and from a traditional activity coefficient calculation is less than 0.5 mV. The concept is validated with an all-solid-state nitrate sensor as well as a commercial fluoride-selective electrode, giving Nernstian responses in different ionic strength backgrounds against concentration without the need for correcting activity coefficients or liquid junction potentials.

EDL Gated FET Biosensor Array for the Investigation of Ion Channels and Bioelectric Signals of Circulating Tumor Cells

This paper demonstrates the applications of novel high sensitive electrical double layer (EDL) gated FET biosensor array in detecting and counting cells, and analyzing their bioelectric signals in different test conditions. We focused on extremely rare cell type such as Circulating Tumor Cells (CTCs) (HCT-8 cell line) derived from Colorectal Cancer (CRC) cell line. CRC is one of the major forms of cancer that affects human population and CTCs have emerged as a clinical biomarker for diagnosis and prognosis of metastatic CRC. Previously, we had shown that our methodology can detect and count CTCs with very high sensitivity, with up to single cell resolution. Simultaneously, we discovered our sensor platform can be used to dynamically monitor the changes in transmembrane potential (Vm) of the cell, which is an important fundamental bioelectric signal of cell. Studies have shown Vm may be useful as a primary parameter in understanding disease progression, cell signaling mechanism and ion channel gating. We demonstrated that the EDL gated FET sensor can dynamically monitor the changes in Vm induced by high concentrations of extracellular KCl. It was also shown that the sensor response changes as extracellular divalent cation concentrations are dynamically increased towards physiological concentrations. The results display the ability of the sensing platform to monitor the cellular response to dynamically changing, very low ion concentrations as well. Furthermore, an interesting application of bioelectric signal study is demonstrated using cadmium ions to block the calcium channels present in the cell. As shown in figure, in low micro molar concentrations, cadmium acts as a permeant channel blocker (electrically, the cell is depolarized and as the Cd2+ concentration increases, it re-polarizes) and in higher concentrations, it accumulates in the intracellular region (the cell is depolarized again), causing induction of apoptotic signals. The sensor response closely follows the cellular response to permeant channel blockers such as cadmium. These results demonstrate that our sensing methodology can replace the conventional electrophysiological methods or optical potentiometric probes to study fundamental cellular mechanisms such as ion channel gating. Our method offers non-invasive means to monitor changes in Vm, with very high sensitivity for individual cells. The cell based studies can be carried out directly in physiological media with high ionic strength, without the use of extensive pre-treatments or additional reagents. Figure 1

Voltammetric pH Nanosensor.

Nanoscale pH evaluation is a prerequisite for understanding the processes and phenomena occurring at solid-liquid, liquid-liquid, and liquid-gas interfaces, e.g., heterogeneous catalysis, extraction, partitioning, and corrosion. Research on the homogeneous processes within small volumes such as intracellular fluids, microdroplets, and microfluidic chips also requires nanometer scale pH assessment. Due to the opacity of numerous systems, optical methods are useless and, if applicable, require addition of a pH-sensitive dye. Potentiometric probes suffer from many drawbacks such as potential drift and lack of selectivity. Here, we present a voltammetric nanosensor for reliable pH assessment between pH 2 and 12 with high spatial resolution. It consists of a pyrolytic carbon nanoelectrode obtained by chemical vapor deposition (CVD) inside a quartz nanopipette. The carbon is modified by adsorption of syringaldazine from its ethanolic solution. It exhibits a stable quasi-reversible cyclic voltammogram with nearly Nernstian dependency of midpeak potentials (-54 mV/pH). This sensor was applied as a probe for scanning electrochemical microscopy (SECM) in order to map pH over a platinum ultramicroelectrode (UME), generating hydroxide ions (OH(-)) by the oxygen reduction reaction (ORR) at a diffusion-controlled rate in aerated phosphate buffered saline (PBS). The results reveal the alkalization of the electrolyte close to the oxygen reducing electrode, showing the insufficient buffer capacity of PBS to maintain a stable pH at the given conditions.

Larry Cohen—50 ways to DYE your science

Larry Cohen—50 ways to DYE your science

Platinized carbon nanoelectrodes as potentiometric and amperometric SECM probes

Micrometer-sized platinized carbon electrodes have previously been used for the detection of reactive oxygen and nitrogen species (ROS and RNS) in biological systems. Here, we report the preparation and characterization of quartz-sealed platinized carbon nanoelectrodes. Such electrodes can be employed as tips in the scanning electrochemical microscope (SECM). The prepared electrodes were characterized by steady-state voltammetry, scanning electron microscopy, and SECM. In addition to ROS/RNS detection, the high surface area of a platinized nanoelectrode makes it a useful potentiometric probe. Unlike previously fabricated platinized electrodes, carbon electrodes possess a very thin insulating sheath, which is essential for experiments inside biological cells and high-resolution SECM imaging.

Effects of marine biofouling on gas sensor membrane materials

The use of underwater gaseous sensors has increased rapidly in the last 10 years. The majority of such sensors employ a thin membrane through which the gas diffuses. These sensors are potentiometric gas-sensing probes and essentially they are ion-selective electrodes. The deployment time of these membranes is curtailed by the formation of biofouling on the membrane leading to erroneous results. The physical properties of a variety of commonly used membranes were investigated using SEM and AFM. This showed that there were differences in topography between the PTFE membranes, such as pore sizes and surface roughness, which may be attributed to the manner in which they are manufactured. The pore size of the PTFE membranes varied greatly, ranging from circular pores with a diameter of 500 nm to elongated pores measuring 1 x 22 microm. The contact angle of each membrane showed that they were all hydrophobic. The amount of fouling on each was also observed and its affect on oxygen diffusion was monitored. Fouling slowed down the response of the instrument and caused reduced diffusion through the membranes. The amount of fouling varied between the membranes with the YSI membrane fouling least. Some of the membranes tested did foul less than others and there could be lifetime advantages of choosing a membrane with a smoother surface and a small pore size.

Fluorescent Probe Based on Intramolecular Proton Transfer for Fast Ratiometric Measurement of Cellular Transmembrane Potential

Development of fast-response potentiometric probes for measuring the transmembrane potential Vm in cell plasma membranes remains a challenge. To overcome the limitations of the classical charge-shift potentiometric probes, we selected a 3-hydroxychromone fluorophore undergoing an excited-state intramolecular proton transfer (ESIPT) reaction that generates a dual emission highly sensitive to electric fields. To achieve the highest sensitivity to the electric field associated to Vm, we modified the fluorophore by adding two rigid legs containing terminal polar sulfonate groups to allow a deep vertical insertion of the fluorophore into the membrane. Fluorescence spectra of the new dye in lipid vesicles and cell membranes confirm the fluorophore location in the hydrophobic region of the membranes. Variation of Vm in lipid vesicles and cell plasma membranes results in a change of the intensity ratio of the two emission bands of the probe. The ratiometric response of the dye in cells is approximately 15% per 100 mV, and is thus quite large in comparison with most single-fluorophore, fast-response probes reported to date. Combined patch-clamp/fluorescence data further show that the ratiometric response of the dye in cells is faster than 1 ms. Analysis of the excitation and emission shifts further suggests that the probe responds to changes in Vm by a mechanism based on electrochromic modulation of its ESIPT reaction. Thus, for the first time, the ESIPT reaction has been successfully applied as a sensing principle for detection of transmembrane potential, allowing to couple classical electrochromic band shifts with changes in the relative intensities of the two well-separated emission bands. The fast two-band ratiometric response as well as the relatively high sensitivity of the new probe are the key features that make it useful for rapid detection of Vm changes in cell suspensions and single cells. Moreover, the new design principles proposed in the present work should allow further improvement of the probe sensitivity.

Assessment of mitochondrial membrane potential in proximal tubules after hypoxia-reoxygenation

Proximal tubules develop a severe energetic deficit during hypoxia-reoxygenation (H/R) that previous studies using fluorescent potentiometric probes have suggested is characterized by sustained, partial mitochondrial deenergization. To validate the primary occurrence of mitochondrial deenergization in the process, optimize approaches for estimating changes in mitochondrial membrane potential (DeltaPsim) in the system, and clarify the mechanisms for the defect, we further investigated the behavior of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazocarbocyanine iodide (JC-1) in these cells and introduce a more dynamic and quantitative approach employing safranin O for use with the tubule system. Although use of JC-1 can be complicated by decreases in the plasma membrane potential that limit cellular uptake of JC-1 and such behavior was demonstrated in ouabain-treated tubules, changes in DeltaPsim entirely accounted for the decreases in the formation of red fluorescent JC-1 aggregates and in the ratio of red/green fluorescence observed after H/R. The red JC-1 aggregates did not readily dissociate when tubules were deenergized after JC-1 uptake, making it unsuitable for dynamic studies of energization. Safranin O uptake by digitonin-permeabilized tubules required very small numbers of tubules, permitted measurements of DeltaPsim for relatively prolonged periods after the end of the experimental maneuvers, was rapidly reversible during deenergization, and allowed for direct assessment of both substrate-dependent, electron transport-mediated DeltaPsim, and ATP hydrolysis-supported DeltaPsim. Both types of energization measured using safranin O in tubules permeabilized after H/R were impaired, but combining substrates and ATP substantially restored DeltaPsim.

Mitochondria in tumor cells studied by laser scanning confocal microscopy.

We present here a confocal fluorescence microscopy study of mitochondria in sensitive and resistant carcinoma cells by using two potentiometric probes of mitochondria, rhodamine 123 (R123) and dimethylaminostyryl-methylpyridiniumiodine. We have found that active mitochondria in sensitive MCF-7 and multidrug resistant MCF-7/DX carcinoma cells are very different in localization and morphology. In sensitive cells active mitochondria are found in the perinuclear region, whereas in the multidrug resistance (MDR) subline they are confined to the cell periphery. Interestingly, the MDR revertant verapamil has been found to restore in MCF-7/DX cells the same pattern of active mitochondria seen in sensitive cells. We have also studied R123 in human lung carcinoma A549 cells, which display a low responsivity to doxorubicin, and overexpress the lung resistance-related protein. In addition to perinuclear mitochondria, peripheral mitochondria with weaker fluorescence can be seen in this cell line. Interestingly, in the two examined carcinoma lines we have been able to recognize by image analysis a common new star-lobed morphology. Our results indicate that in resistant carcinoma cells two populations of mitochondria coexist with different localization, morphology, and activity.

Novel hyperpolarizable and fluorescent dyes in lipid membranes: studying membrane potentials using nonlinear optical and fluorescence

Potentiometric optical probes gain importance. Here, we report on the application of those probes to study transmembrane potentials using nonlinear optical (NLO) and fluorescence techniques. A NLO spectroscopy method is presented which allows for precise recording of membrane potential changes. To this end the signal intensity of second harmonic generation (SHG) mediated by novel acetylenic hemicyanine dye molecules embedded in the membrane of Retzius neuron cells was used. Bichromophoric systems have been designed for efficient energy transfer measurements. In many cells these bichromophores change their behaviour from stationary fast-response dyes to slow-response redistribution dyes. Completely novel potentiometric probes acting in a different and most efficient way were constructed using one aminostyrylpyridinium (ASP) bichromophoric dye and one oxonol dye. The redistribution ability of the ASP dye can be optimised by controlled and tailor-made modification of its substituents. In combination with the anionic oxonol dye DiBAC 4(3) a bichromophoric system results with two chromophores showing opposite redistribution behaviour. Fluorescence ratio changes of 100% for a 100 mV depolarization can be recorded.

Coupling of Simultaneous Fluorescence and Electrophysiology: Historical Survey, Contributions, and Prospects

The coupling of simultaneous fluorescence and eleclrophysiological measurements paved the way for the development of potentiometric and intracellular free ion-sensitive probes, although the basic and initial aim of these combined studies remains to record fast conformational changes during ion channel activation. Recent high-resolution studies of some channels put back on the agenda challenging methods that combine spectroscopy and electrophysiology. Fluorescence is certainly the most versatile and sensitive spectroscopy in this kind of experiment, and we have recently witnessed significant breakthroughs, at the level of whole cells with intact or mutated channels or with planar lipid bilayers doped with channels or their peptide models. After our initial study of membrane dynamics associated with excitability, following transient pyrene excimer signals during action potentials or voltage-clamp of nerve fibers, we tested the feasability of FRAP (fluorescence recovery after photobleaching) experiments on planar lipid bilayers. This technique was later applied to lateral diffusion measurements of channel forming peptides (alamethicin labeled with fluorescein and the voltage-sensitive segment S4 of the Shaker potassium channel labeled with NBD) under applied voltage. We provide independent evidence for voltage-dependent partitioning and transmembrane insertion and propose renewed experimental avenues to reveal movements and conformational changes associated with ion channel gating and opening.