Membrane potential is the difference in voltage between the interior and exterior of a cell. The membrane potential allows a cell to function as a battery, providing power to operate a variety of molecular devices embedded in the membrane. In electrically excitable cells such as neurons, membrane potential is used for transmitting signals between different parts of a cell. Opening or closing of ion channels at one point in the membrane produces a local change in the membrane potential, which causes electric current to flow rapidly to other points in the membrane. Ion channels have been identified as important drug discovery targets.

The plasma membrane of a cell typically has a transmembrane potential of approximately –70 mV (negative inside) as a consequence of K+, Na+ and Cl–concentration gradients that are maintained by active transport processes. Potentiometric probes offer an indirect convenient method of detecting the translocation of these ions although the fluorescent ion indicators can be used to directly measure changes in specific ion concentrations. Potentiometric optical probes enable researchers to perform membrane potential measurements in organelles and in cells that are too small for microelectrodes. Moreover, in conjunction with imaging techniques, these probes can be employed to map variations in membrane potential across excitable cells, in perfused organs and ultimately in the brain in vivo with spatial resolution and sampling frequency that cannot be obtained using microelectrodes.

Increases and decreases in membrane potential play a central role in many physiological processes, including nerve-impulse propagation, muscle contraction, cell signaling and ion-channel gating. Potentiometric probes are important tools for studying these processes, as well as for visualizing mitochondria (which exhibit transmembrane potentials of approximately –150 mV, negative inside matrix), for assessing cell viability and for high-throughput screening of new drug candidates.

Potentiometric probes include the cationic or zwitterionic styryl dyes, the cationic carbocyanines and rhodamines, and the anionic oxonols. The class of dye determines factors such as accumulation in cells, response mechanism and toxicity. Selecting the best potentiometric probe for a particular application can be complicated by the substantial variations in their optical responses, phototoxicity and interactions with other molecules. There are two classes of membrane potential probes based on their response mechanisms: fast response and slow response membrane potential dyes.

Fast-response probes have their fluorescence in response to a change in the surrounding electric field. 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 the redistribution of the fluorescent anion is from millisecond to second, which is in the time scale for measuring the rapid cellular electrical events. 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 are primarily sensitive to plasma membrane potential. Potential-dependent fluorescence changes generated by DiBAC4 (3) are typically ~1% per mV.