The development of a fiber-optic probe for the in vivo resonance Raman spectroscopy of neurotransmitters

The measurement of neurotransmitter secretions by living cells, both in living organisms or in preparations, constitutes an enduring and vexing problem for neuroscientists due to the large number of substances involved at very low concentrations. An ability to correlate neurotransmitter secretions with various factors including organismic behavior would greatly advance our understanding of the organization and functioning of central nervous systems. This, in turn, has many important implications for the diagnosis and treatment of disorders of central nervous systems (mainly in humans) as well as for the design and implementation of information processing and control systems. The work presented here was undertaken in order to explore a novel approach to this demanding problem. The objective was to develop a probe capable of measuring neurotransmitter secretions in real time, at physiologically relevant concentrations, and non-invasively in situ. Data were obtained using an ultraviolet resonance Raman spectroscopic analytical technique performed via optical fibers, and were analyzed primarily with artificial neural networks. To this end, a prototype tunable ultraviolet resonance Raman system was designed, assembled, comissioned and employed. A general introduction to the problem and a discussion of existing techniques for neurotranmitter measurement are given in Part I. In Part n, the analytical method was shown to allow discrimination between several different neurotransmitters and some of their precursors, both on the basis of their spectra and the selective resonance enhancement of their spectra. Optical fibers were characterized with regard to their suitability for use with pulsed ultraviolet radiation in Part III and on the basis thereof selected for the construction of optical fiber probes. It was found that the performance of optical fibers varied greatly when subjected to pulsed ultraviolet radiation, making the selection of fibers a crucial factor in probe construction. Various design features influencing the efficiency of optical fiber probes were investigated using both theoretical and empirical techniques. A right-angle geometry using a small diameter excitation fiber and several larger collection fibers in close proximity produced the most efficient probe. In Part IV the use of cell secretions as samples modelling in vivo conditions were investigated. It was also shown that these probes could be inserted via surgically implanted cannulae into and operated in the crania of experimental male rats without producing discernable behavioral artifacts. In Part V some signal recovery methods were investigated and it was shown that artificial neural networks could be used to identify and quantify neurotransmitters based on their Raman spectra. Part VI contains an assessment of the neuroprobe using neurotransmitter secreting cultured cells as a model system. The thesis is concluded with a discussion of the charateristics of an ideal biosensor, reviews the work done, and highlights some future directions. This thesis represents my contributions toward the development of a tunable ultraviolet resonance Raman neurotransmitter probe. Within the scope of this work, limitations of the available equipment and other resources precluded the complete development of a high-performance neuroprobe, however, the data presented here demonstrate proof-of-concept and feasibility. In particular, what has hitherto been considered impossible - the use of optical fibers for pulsed ultraviolet remote resonance Raman spectroscopy - has been shown to be distinctly feasible. It has further been shown that ultraviolet resonance Raman spectroscopy is well-suited to the problem of resolving a mixture of neurotransmitters in a biological matrix. With the appropriate state-of-the-art equipment, there is now a very real possibilty of obtaining detection limits of lx10~9 M for the catecholamine neurotransmitters and 1x10"0" M for the aliphatic neurotransmitters with 30 s exposure time, thus providing a novel and general solution to the problem of neurotransmitter measurement.Graduate and Postdoctoral StudiesGraduat