This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. As part of the BRC's role as an integrated technology resource, we strive to bring together different experimental approaches, providing parallel data collection from single cells. This effort encompasses hybrid sensors, single microelectrodes combining electrochemical and optic approaches, as well as the construction of integrated platforms. Hybrid Sensors Ion-selective electrodes used in the self-referencing configuration (SERIS probes) are highly sensitive, noninvasive detectors of slow diffusion gradients of ions such as protons or calcium from epithelial sheets or even from single cells. The molecular mechanisms of secretion, however, are usually controlled from within the cell, a domain into which a conventional ion-selective electrode cannot penetrate. To gain simultaneous information about the amount of ion secretion and the mechanisms controlling secretion, a dual probe is under construction. The dual probe consists of an ion-selective electrode with a fiber-optic light carrier inserted through the tip, for delivery of excitation light to an arbitrary reporter molecule. A prototype probe has been constructed, following resolution of several technical issues. To avoid destroying the selectivity of the ionophore, the optical fiber had to be silanized. After silanization, selectivity of the ionophore was unchanged, as measured with both calcium- and proton-selective ionophores. Coupling of excitation light into the optical fiber required vibrating the fiber loading end at high (20 kHz) frequencies, to ensure even delivery of optical modes and reduce laser speckle. The microscope was equipped with fluorescein-based filters to allow delivery of blue laser light (argon ion laser) and detection of green emission light. Integrated Platforms New technologies are more generally accepted when they are combined with existing techniques operating in parallel and provide mutual corroboration. The BRC already maintains combined electrochemical and electrophysiological platforms, the later encompassing single microelectrode, voltage, current and patch clamp.
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