Quantitative analysis of membrane potentials.

The changes that occur in electrochemical gradients across biological membranes provide us with invaluable information on physiological responses, pathophysiological processes and drug actions/toxicity. This chapter aims to provide researchers with sufficient information to carry out a quantitative assessment of mitochondrial energetics at a single-cell level thereby providing output on changes in the mitochondrial membrane potential (Deltapsi(m)) through the utilization of potentiometric fluorescent probes (TMRM, TMRE, Rhodamine 123). As these cationic probes behave in a Nernstian fashion, changes at the plasma membrane potential (Deltapsi(p)) need also to be accounted for in order to validate the responses obtained with Deltapsi(m)-sensitive fluorescent probes. To this end techniques that utilize Deltapsi(p)-sensitive anionic fluorescent probes to monitor changes in the plasma membrane potential will also be discussed. In many biological systems multiple changes occur at both a Deltapsi(m) and Deltapsi(p) level that often makes the interpretation of the cationic fluorescent responses much more difficult. This problem has driven the development of computational modelling techniques that utilize the redistribution properties of the cationic and anionic fluorescent probes within the cell to provide output on changes in Deltapsi(m) and Deltapsi(p).