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 ref and ultimately in the brain in vivo ref with spatial resolution and sampling frequency that cannot be obtained using microelectrodes.

Applications for Potentiometric Probes

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 method of detecting the translocation of these ions, whereas the fluorescent ion indicators discussed in Indicators for Na+, K+, Cl– and Miscellaneous Ions—Chapter 21 can be used to directly measure changes in specific ion concentrations.

Increases and decreases in membrane potential—referred to as membrane hyperpolarization and depolarization, respectively—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) (Probes for Mitochondria—Section 12.2), for assessing cell viability (Viability and Cytotoxicity Assay Reagents—Section 15.2) and for high-throughput screening of new drug candidates.

Potentiometric probes include the cationic or zwitterionic styryl dyes, the cationic carbocyanines and rhodamines, the anionic and hybrid oxonols and merocyanine 540. The class of dye determines factors such as accumulation in cells, response mechanism and toxicity. Surveys of techniques and applications using membrane potential probes can be found in several reviews.ref

Selecting a Potentiometric Probe

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. Probes can be divided into two categories based on their response mechanism:

  • Fast-response probes (usually styrylpyridinium dyes, Fast-Response Probes—Section 22.2) operate by means of a change in their electronic structure, and consequently their fluorescence properties, in response to a change in the surrounding electric field (Figure 22.1.1). 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; fast-response probes typically show a 2–10% fluorescence change per 100 mV.
  • Slow-response probes (Slow-Response Probes—Section 22.3) exhibit potential-dependent changes in their transmembrane distribution that are accompanied by a fluorescence change (Figure 22.1.1). 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.

Calibration of potentiometric probes can be accomplished by imposing a transmembrane potential using valinomycin or gramicidin (V1644, G6888; Fluorescent Na+ and K+ Indicators—Section 21.1) in conjunction with externally applied K+ solutions.ref The ultimate test of calibration veracity is quantitative agreement with electrophysiological measurements.ref

Mechanisms of Membrane Probes

Figure 22.1.1 Response mechanisms of membrane potential–sensitive probes. Fast-response probes undergo electric field–driven changes of intramolecular charge distribution that produce corresponding changes in the spectral profile or intensity of their fluorescence (represented by color changes in the illustration). Slow-response probes are lipophilic anions (in this illustration) or cations that are translocated across membranes by an electrophoretic mechanism. Fluorescence changes associated with transmembrane redistribution (represented by color changes in the illustration) result from sensitivity of the probe to intracellular and extracellular environments. Thus, potentiometric response speeds directly reflect the time constants of the underlying processes—fast intramolecular redistribution of electrons versus relatively slow transmembrane movement of entire molecules.

For Research Use Only. Not for use in diagnostic procedures.