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