UC San Diego

Understanding cellular behavior and tissue organization requires a deeper understanding of nanoscale structure and activity of proteins and biomacromolecules, including channels and receptors that act as messengers connecting the cell to its surrounding. Channels and receptors respond to a wide variety of electrical, chemical, and mechanical stimuli to facilitate cellular homeostasis, communication, migration, and survival. Ion channels facilitate the passage of ions and metabolites across cellular membranes and are visualized by high-resolution 3D imaging techniques, which include EM and scanning probe microscopies, such as atomic force microscopy (AFM) and scanning ion conductance microscopy (SICM). However, the current imaging techniques are unable to obtain the intertwined direct relationships between structure and electrical activity of ion channels. My work is dedicated to designing and implementing such an integrated system. This dissertation describes the details about different novel AFM-based nanotechnologies designed and developed for simultaneous structure-activity imaging of various electrically active biological systems. First, a novel AFM probe was developed by insulating tungsten micro-wires, which can measure electrical activity at the nanoscale. These probes, coated in gold, were used to image the structure of Escherichia coli that surface express mutants of the redox active enzyme, alcohol dehydrogenase II. Simultaneous structure-function imaging of the bacteria cells revealed improved electron transfer when mediators were placed closer to the NADH binding pocket. Second, a two-chamber system mimicking biological membranes (~5 nm thick) that enables the imaging of ion channel proteins in lipid membrane models was developed. The two chambers were separated by a 5 nm thick insulated graphene sheet deposited over a 1 μm hole. A TEM was used to drill a ~20 nm pore. The substrate supports lipid membranes for measuring electrical activity. Third, AFM was used to image cell-surround communication channels (Connexin26 hemichannels) in purified membrane plaques as well as in reconstituted lipid membranes revealing channel clustering in high-resolution images. The electrical activity of these hemichannel preparations were then recorded when de- posited over the nanopore supports for initial simultaneous electrical recording and imaging. Lastly, the design and development of a parallel SICM-array capable of simultaneous multi-point high-throughput nanoscale imaging was realized.