A microscope system will be constructed that can generate image contrast from the Raman scattering of water inside living cells; it will then be used to measure water flux between intracellular compartments. Although water is the most abundant molecule inside cells, and the solvent of nearly all intracellular chemistry, the mechanisms that control its intracellular distribution are unknown. Its dynamics across the plasma membrane must be inferred by indirect methods, which are not easily applied to intracellular organelles. The Swanson lab has studied movements of intracellular water in macrophages through studies of fluid phase pinocytosis and nuclear structure. It has developed quantitative, fluorescence microscopic methods for the study of fluid movements inside cells. For his doctoral thesis in chemistry, Dr. Christensen designed and built several microscopes that generated image contrast using Raman spectral bands from a variety of compounds, including water. The principles of that technology will now be incorporated into a microscope that can rapidly acquire water Raman images of living cells. The Raman microscope will then be used to measure water flux between cytoplasm and vesicular intracellular compartments that are essential for osmoregulation or endocytosis. The project will consist of two phases. In year 1, a microscope and laser will be purchased and configured with present lab equipment to collect Raman signals from H2O and D2O in cells. In year 2, an intensified, cooled CCD camera, and additional microscope components will be purchased to provide more sensitive acquisition of Raman images from cells during a rapid exchange of solvents (H20 and D20). These methods will be used to measure water exchange across membranes of pinosomes, phagosomes and contractile vacuoles inside living cells. Eventually, this microscope could be used to detect Raman signals from other abundant intracellular molecules, and to measure additional parameters obtainable from water structure, such as temperature and pH.
