Development and characterization of an optical fiber based instrument for ultraviolet resonance Raman spectroscopy of biomolecules

Proteins are involved in virtually all natural biological processes, as well as many industrial and clinical applications. The function and activity of a protein are determined by its primary, secondary, tertiary and quaternary structures which dictate how it interacts with other biomolecules and its environment. Determination of protein structure is critical in elucidating mechanisms of protein action and in understanding protein behaviour, and protein-mediated processes and functions. A number of spectroscopic techniques are common for protein structure determination, the choice of which is often determined tradeoffs between (a) level of stucture required, (b) performance, (c) adaptability to in situ and/or in vivo use, and (d) cost. There exists a need for an inexpensive instrumental technique with moderate sensitivity to secondary and tertiary structure and an ability to operate remotely (in vivo and in situ). This document describes the design, development and application of the first fiber-optic linked instrument for ultraviolet resonance Raman spectroscopy (FO-UVRRS) which meets these criteria. A distinctly systems engineering approach to this problem was adopted. Starting with a definition of the problem and design criteria (Chapter 1), design of the FO-UVRRS system proceeded by considering the relationship between the major hardware components (Chapter 2). In optimizing the system, two major problems were encountered which resulted in detailed investigations: (a) stable transmission of high intensity ultraviolet light through new silica optical fibers without catastrophic bulk/surface damage or colour-centre induced solarization (Chapter 3), and (b) characterization and optimization of fiber-optic probe design for use in situ with highly absorbing samples (Chapter 4). Specialized signal-to-noise ratio enhancement techniques were investigated as a further means of improving the system (Chapter 5). The efficacy of the probes was demonstrated through applications to systems of biological import (Chapter 6), including specific and non-specific protein binding. These demonstrations comprise the first reported fiber-optic linked biophysical spectroscopic investigations at deep UV wavelengths and represent a significant contribution to biomolecular spectroscopy. Collectively, the research described here has resulted in novel designs, mathematical models, and optical materials and biophysical data which are immediately useful for UVRRS instrument design as well as other future applications.Applied Science, Faculty ofElectrical and Computer Engineering, Department ofGraduat