Optical, electrical and electrochemical techniques for biophysical studies of single, excitable cells

Established technologies such as amperometry and electric impedance spectroscopy (EIS) have been used with great success in studies of cellular function and cellular pathophysiology, as well as in early-stage detection of disease. However, the application of these tools, particularly at the single-cell scale, can be technically prohibitive. The focus of this dissertation has been on the development of techniques to facilitate routine application of these methods in biophysical studies of single, excitable cells. In the first phase of this research, we designed, fabricated and characterized a microchip platform that allowed for subcellular, fringe-field EIS measurements of cell membrane electrical properties, along with recordings of cellular exocytosis. Initial results demonstrated the ability of the microchip to coarsely resolve intracellular structures of isolated cochlear outer hair cells as well as quantal neurotransmitter release events from rat pheochromocytoma (PC12) cells. Using this platform, in the second phase, we introduced a technique for microchip-based extracellular electrical stimulation of cells and simultaneous amperometric detection of catecholamine release. Specifically, results with PC12 cells demonstrated that the electric field generated by the potentiostat used for electrochemical detection would automatically trigger cellular exocytosis. We showed that this technique retained the high sensitivity of conventional amperometry in resolving single vesicle quantal release events and even the occasional fusion pore formation event. However, beyond the traditional approach, results indicated detection of release from remote sites around the cell and the potential for higher-throughput, exocytosis flow cytometry. In the final phase of this dissertation, we performed experiments to examine the origins of pulsed infrared (IR)-evoked cell excitability as a potential tool for chip-integrable, rapid, artifact-free and reversible stimulation of cells. Fluorescence confocal microscopy of the cells revealed that IR irradiation triggered cellular excitation through the generation of intracellular calcium release and uptake events. Pharmacological testing implicated mitochondria as the major component of the IR-evoked responses. Overall, the research in this dissertation provides contributions in the development, elucidation and application of new technologies that facilitate single cell biophysical studies.