Use of Impedance Spectroscopy As A Probe of Mitochondrial Membrane Potential

A key element in mitochondrial metabolism, the synthesis of adenosine triphosphate (ATP), is driven by an electrochemical potential and a proton concentration gradient across the inner mitochondrial membrane. The electrical properties of mitochondrial suspensions thus provide vital information into internal metabolic processes. This study reports on measurements of frequency- and time-dependent electrical impedance of mitochondrial suspensions as a non-invasive and label-free method of probing mitochondrial membrane potential, and other aspects of mitochondrial activity. We designed and fabricated an effective and sensitive probe to study changes in electrical properties of live mitochondrial suspensions involving basic bioenergetic respiratory protocols, to monitor the activity of the electron transport chain. Several substrates, inhibitors, and uncouplers were used to stimulate the electron transport mechanism in mitochondria, and study the oxygen consumption changes. These observed oxygen consumption changes were correlated with measured impedance changes of mitochondrial suspensions of various concentrations, by studying their dielectric properties. Our measurements of complex impedance were used to calculate relative dielectric permittivity and conductivity of mitochondrial suspensions, and to obtain admittance data that allowed analysis of the effects of interactions between substrates and mitochondria. Our results indicate that changes in relative dielectric permittivity for mitochondria in the presence of pyruvate and malate correlate with the increased oxygen intake of mitochondria. Our findings show that these two substrates, which trigger activation of the electron transport chain and increase the membrane potential, are shown to increase the effective relative dielectric permittivity, correlating with the expected membrane potential change. Thus, an impedance sensing method can be adapted for in-vitro studies of biological processes, such as those that take place within mitochondria. The method discussed in this dissertation could eventually be developed into tools that monitor mitochondrial diseases and dysfunction, and help enable development of treatment methods, such as new drugs that target metabolism.