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 recombinant systems comprised of highly unsaturated lipids and/or a variety of saturated lipids and an appropriate integral membrane protein. Established order/disorder parameters pertinent to each lipid class and to each chain system are determined, as well as a quantization of the formation of lipid microclusters. An understanding of the sizes and formation of fluctuating membrane microclusters allows an examination of the effects of lipid microdomains on protein conformational rearrangements. Specifically, using infrared spectroscopic techniques, we examined in detail the lipid control of both the photocycle activity of bacteriorhodopsin and the conformational flexibility of the protein's integral membrane alpha helices. (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 our studies involving large numbers of prostate tissue sections in the form of tissue microarrays in which the vibrational spectral signatures for control, prostatic intraepithelial neoplastic and tumor tissues were examined. In this case, our imaging instrumentation incorporated highly sensitive linear array detection for rapidly recording hypercube spectral data. For spectroscopically elucidating the various histological features present in prostate tissue, extraordinarily large spectral training sets and appropriate spectroscopic metrics were developed for distinguishing between ten morphological entities occurring in prostatic tissue. This approach is currently being developed for ascertaining prostatic adenocarcinoma, in particular, and for application to other tissue pathologies. With regard to the infrared imaging instrumentation, a number of enhancing features were made in the optics, in detector configurations, and in data collection paradigms. In particular, we have implemented a generalized form of interferometric rapid-scan infrared spectroscopic imaging (to be distinguished from interferometer step-scan approaches) for utilization with any type of focal plane array detector. Further, we are the first group to implement time-resolved Fourier-transform infrared spectroscopic imaging which permits, for example, the visualization of repetitive dynamic processes with half lives on the order of milliseconds. The example used in this case involved specific polymer liquid crystal composites. (2B) Our imaging approaches have also been extended to the visible spectral region in which reflectance spectra are obtained using CCD detection and appropriate liquid crystal tunable filters for wavelength discrimination. This particular noninvasive reflectance unit has been used successfully in clinical venues for determining tissue oxygenation in patients with sickle cell disease in which we examined the effects of nitric oxide stimulation, inhibition and administration. We concluded that patients with sickle cell disease exhibit impaired tissue oxygenation despite having resting blood flow values that were two-fold higher than healthy African American subjects. Furthermore, when pharmacologically increasing blood flow by seven-fold, tissue oxygenation was improved, but remained well below healthy subjects. This versatile visible reflectance imaging approach suggests a useful, real time probe of patients with vascular disease that allows a novel means for assessing disease severity and disease progression.
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