| Description |
Subcellularly resolved, excitable changes (i.e., those induced by electrical or chemical stimuli) in membrane capacitance, influenced by factors including integralmembrane protein activity, lipid densities and membrane-bound water content, may be used to elucidate nonconductive ion-channel conformational state changes, lipid-raft locations and drug-membrane binding processes. However, membrane capacitance has proven difficult to measure, partially because of bandwidth limitations associated with glass/quartz pipettes used during conventional electrophysiology. To address these challenges, techniques introduced in this thesis integrate the principles of extracellular radio frequency (RF) recording with conventional two-electrode voltage clamp (TEVC) to 1) spatially resolve effective membrane capacitance and 2) monitor excitable changes in effective membrane capacitance. Furthermore, this thesis also introduces a new multielectrode method to approximate electrode-electrolyte interfacial impedance, which might prove useful in electric impedance spectroscopic or electric impedance tomographic applications. Specific contributions include the following: 1) A method that simultaneously estimates double-layer and interelectrode (chamber) impedances, in the linear regime of electrode voltage-current sensitivity, during extracellular electrode-based measurements. This method estimates impedance parameters by applying a nonlinear least-squares regression to measurements between various groups or pairs of a three-electrode system and, unlike previous double-layer approximation methods, can be done without the use of multiple calibration solutions or moveable electrode configurations. 2) A platform capable of visualizing the spatial distribution of membrane capacitance, using extracellular RF electrode recordings, around a single cell. The proof-of-concept for this technique is demonstrated with dielectric maps around polarized Xenopus oocyte membranes. 3) Development and characterization of a platform to enable RF impedancebased measurements around voltage-clamped ShakerB-IR-expressing Xenopus oocytes. Data indicated that the platform was most sensitive to effective changes in oocyte dielectric at 300 kHz and 500 kHz. 4) Temporal characterization of changes in voltage-sensitive RF membrane capacitance associated with ShakerB-IR activation (expressed in Xenopus oocytes) and ShakerB-IR-Cu2+ interactions. Results indicate that extracellular RF-impedance-based measurements can temporally and spatially elucidate changes in excitable cell-membrane capacitance and could supplement conventional electrophysiological techniques to provide a broader understanding of cellular biophysics. |
