Summary of Work: Our research efforts encompassed two general areas: (1) The modulatory effects of bilayer lipids on the structural reorganizations of integral membrane proteins, and (2) the instrumental development and applications of vibrational Raman and infrared spectroscopic imaging techniques. (1) Our interest in characterizing the effects of fluctuating lipid microdomains within biomembranes has recently focused on cluster formation within bilayer matrices comprised of lipid mono- or polyunsaturated sn-2 chain and saturated sn-1 chain assemblies. The lateral compressibility properties of these lipid microaggregates are effective in exerting a modulatory influence on induced conformational changes occurring within integral membrane proteins. In studying spectroscopically specific lipid bilayers, appropriate acyl chain deuteration allows the vibrational dynamics of each chain moiety to be monitored separately. We have continued the utilization of both Raman and infrared spectroscopic techniques, in conjunction with freeze-quenching methodologies, toward examining model bilayer systems comprised of highly unsaturated lipids, as, for example, didocosahexaenoylphosphatidylcholine, DDPC, and fully deuterated disaturated acyl chain lipids, namely, dipalmitoylphosphatidylcholine, DPPC, and disteroylphosphatidycholine, DSPC. Established order/disorder parameters pertinent to each lipid class and to each chain system were determined, as well as a quantization of the formation of lipid microclusters. An understanding of the sizes and formation of fluctuating membrane microclusters will allow us to pursue the next step in which rhodopsin, an integral membrane protein, is inserted into the bilayer and the effects of lipid microdomains on protein conformational rearrangements are examined. (2A) Considerable emphasis has been placed on enhancing our mid-infrared spectroscopic chemical imaging microscopy techniques by combining step-scan interferometry with state-of-the-art infrared sensitive two-dimensional focal plane array detectors. The integration of high performance digital imaging with noninvasive, high resolution optical spectroscopy allows a visualization of the spatial distribution of distinct chemical species in a variety of host environments. The power of the technique is also manifest in the simultaneous acquisition of an infrared spectrum for each spatial location. As an example of the utility of the infrared imaging technique in diagnostic pathology, promising results were obtained from prostate tissue sections in which the vibrational spectral signatures for control, prostatic intraepithelial neoplastic and tumor tissues exhibited demonstrable differences. With regard to the infrared imaging instrumentation, a number of enhancing features were made in the optics, as well as in detector configurations. We have developed a novel data collection technique for step-scan, microimaging spectrometers that allows large numbers of, for example, histological samples to be imaged rapidly with a high signal-to-noise ratio (SNR). We have proposed a theoretical description for the performance characteristics of infrared hyperspectral imaging systems and have also explained theoretically the quantitative effects of the acquisition parameters on the SNR. Further, the theoretical analysis is extended to Fourier transform infrared micro-imaging employing continuous scan interferometers, a new development. For data acquisition and processing protocols we have modified gain ranging theory to account for the variation of noise by incorporating a linear model for noise prediction. A "median filtered time averaging" method for effectively reducing noise in Fourier transform imaging data has been rigorously discussed and introduced. (2B) Our imaging approaches have also been extended to the visible spectral region in which reflectance spectra are obtained using a CCD detector and appropriate liquid crystal tunable filters for wavelength discrimination. This particular noninvasive reflectance unit has been used successfully in the clinic for assessing hemoglobin saturation during systemic nitric oxide (NO) inhibition and subsequent nitric oxide inhalation. These studies demonstrate that a significant decline in the percentage of oxyhemoglobin occurs in skin tissue when blood flow is reduced after inhibition of forearm NO synthesis and that there is a restoration of oxyhemoglobin toward basal values with improved blood flow during inhalation of NO. This versatile imaging approach suggests a useful, real time probe of patients with vascular disease.