Membrane potential is the difference in voltage between the interior and exterior of a cell. It allows cells to function as electrochemical systems that regulate the activity of membrane-embedded proteins, including ion channels and transporters. In electrically excitable cells such as neurons and muscle cells, changes in membrane potential underlie signal propagation, where opening or closing of ion channels produces local voltage changes that spread along the membrane. Because ion channels regulate these electrical signals, they represent important targets in drug discovery.

The plasma membrane of most cells maintains a resting membrane potential of approximately –70 mV (negative inside), which arises primarily from potassium ion permeability and is sustained by sodium, potassium, and chloride gradients established by active transport processes such as the Na⁺/K⁺-ATPase. Potentiometric probes provide an indirect method for detecting changes in membrane potential resulting from ionic fluxes, whereas fluorescent ion indicators directly measure changes in the concentration of specific ions. Potentiometric optical probes enable membrane potential measurements in cells and subcellular compartments that are inaccessible to microelectrodes and, when combined with imaging techniques, allow spatial mapping of voltage changes in excitable tissues.

Changes in membrane potential play central roles in physiological processes including nerve impulse transmission, muscle contraction, cell signaling, and ion-channel gating. Certain classes of potentiometric probes are also used to assess mitochondrial membrane potential, cell viability, and ion-channel activity in high-throughput screening applications. Potentiometric dyes include styryl dyes, carbocyanines, rhodamines, and oxonols, which differ in charge, response mechanism, cellular localization, and toxicity. Based on their response kinetics, membrane potential probes are broadly classified as fast-response dyes, which report rapid voltage changes, and slow-response dyes, which report steady-state or averaged membrane potential changes.

Fast-response probes change their fluorescence properties through electrochromic mechanisms in response to alterations in the surrounding electric field across the membrane. Their optical response is sufficiently fast to detect transient (millisecond) potential changes in excitable cells, including single neurons, cardiac cells and intact brains. However, the magnitude of their potential-dependent fluorescence change is often small, typically 0.02–0.1% fluorescence intensity change per mV.

The ANEP dyes are among the most sensitive of the fast-response probes. They are essentially nonfluorescent in aqueous solutions and exhibit spectral properties that are strongly dependent on their environment. When bound to phospholipid vesicles, di-8-ANEPPS has absorption/emission maxima of ~467/631 nm as compared with ~498/713 nm in methanol. Di-8-ANEPPS responds to increases in membrane potential (hyperpolarization) with a decrease in fluorescence excited at approximately 440 nm and an increase in fluorescence excited at >530 nm. These spectral shifts permit the use of ratiometric methods to correlate the change in fluorescence signal with membrane potential. Zwitterionic di-8-ANEPPS exhibits fairly uniform 10% per 100 mV changes in fluorescence intensity in a variety of tissue, cell and model membrane systems. The millisecond-range temporal characteristics of the ANEP dyes compensate for this modest response amplitude.

RH dyes are predominately used for functional imaging of neurons. There is no a single dye that can provide the optimal response under all experimental conditions. Currently, the most widely used RH dyes are RH 237, RH 421 and RH 795. Like the ANEP dyes, the RH dyes exhibit varying degrees of fluorescence excitation and emission spectral shifts in response to membrane potential changes. Their absorption and fluorescence spectra are also strongly dependent on the environment. Spectra of RH 414 bound to phospholipid vesicles are similar to those obtained on neuronal plasma membranes. The RH dyes (e.g., RH 237) might be used in conjunction with fluorescent Ca²⁺ indicators (e.g., Rhod-2 AM and Rhod-4™ AM) for the simultaneous optical mapping of membrane potential and intracellular calcium in cardiomyocyte monolayers.

Slow-response probes exhibit potential-dependent changes in their transmembrane distribution that are accompanied by a fluorescence change. The magnitude of their optical responses is much larger than that of fast-response probes, typically a 1% fluorescence change per mV. Slow-response probes, which include cationic carbocyanines and rhodamines and anionic oxonols, are suitable for detecting changes in average membrane potentials of nonexcitable cells caused by respiratory activity, ion-channel permeability, drug binding and other factors.

The cationic carbocyanine dyes accumulate on hyperpolarized membranes and are translocated into the lipid bilayer. Aggregation within the confined membrane interior usually results in decreased fluorescence, although the magnitude and even the direction of the fluorescence response are strongly dependent on the concentration of the dye and its structural characteristics. DiOC6 (3) and DiOC5 (3) have been the most widely used carbocyanine dye for membrane potential measurements. In flow cytometry measurements, the detected intensity of carbocyanine fluorescence is dependent not only on the membrane potential, but also on cell size. In some cases, measurements of forward light scatter have been used to normalize the optical changes for cell size variability.

The anionic bis-isoxazolone oxonols accumulate in the cytoplasm of depolarized cells by a Nernst equilibrium–dependent uptake from the extracellular solution. Their voltage-dependent partitioning between water and membranes is often measured by absorption rather than fluorescence. Oxonol VI gives the largest spectral shifts, with an isosbestic point at 603 nm. In addition, oxonol VI responds to changes in potential more rapidly than oxonol V.

DiBAC and DiSBAC dyes are usually ionized, hydrophobic and cell-permeable. When incubated with cells, these hydrophobic probes are intended to move from the aqueous phase (culture medium) to the lipid phase (plasma membrane). The negatively charged inner side of a resting cell membrane prevents the further movement of these ionized dyes. Upon the depolarization of the membrane potential, they translocate to the interior of the cells where they bind to intracellular proteins or lipids that enhance DiBAC and DiSBAC fluorescence. Typically, the time constant for redistribution of the fluorescent anion occurs on the order of seconds, making DiBAC and DiSBAC dyes suitable for monitoring slow or steady-state changes in membrane potential rather than rapid electrical events such as action potentials. Increased depolarization results in more influx of the anionic dye and thus an increase in fluorescence. Conversely, hyperpolarization is indicated by a decrease in fluorescence. In contrast to cationic carbocyanines, anionic DiBAC and DiSBAC2 (3) dyes are largely excluded from mitochondria and function primarily as indicators of plasma membrane potential rather than mitochondrial membrane potential. Potential-dependent fluorescence changes generated by DiBAC4 (3) are typically ~1% per mV.