This application is the continuation of the efforts, started three years ago in our laboratory, to develop the technique of Differential Polarization Microscopy and it applications to various structural problems of biomedical significance. During the last three years, we have built two different prototypes of a differential polarization microscope. The first one uses an image dissector camera, and the second, using confocal optics, represents a substantial improvement over the first. These instruments have been tested and have been used to demonstrate the power of this technique in the structural elucidation of complex sub-cellular structures. Parallel to these developments, we have derived the theory of measurement of this imaging technique. The expressions obtained have been used to extract quantitative structural information from the systems studied. WE have also initiated the use of this method to study the higher order structure of chromosomes, and carried out the experimental and theoretical characterization of the optical activity signals associated with the process of DNA condensation in-vitro. In this application, we propose a number of studies whose ultimate goal is to elucidate the mechanism and process of folding of chromatin along the cell cycle. To this end, we propose to combine the spectroscopic and analytical power of differential polarization microscopy with the use of a recently developed fluorescence microscopy technique. Differential polarization imaging will be used to follow in-situ, the changes in chirality undergone by chromatin along the cell cycle, and to study the higher order organization of metaphase chromosomes. The fluorescence microscopy technique will be used to follow, in real-time, the actual process of condensation of individual fibers of chromatin and single molecular of DNA, under control conditions. The experiments proposed here possess a hierarchy of complexity. The in- vitro studies of single DNA molecule condensation induced by poly-lysine, will be used as a framework to interprete and analyze the results of the studies designed to follow, with the same method, the salt-induced condensation of chromatin in-vitro. Finally, these studies will also be used as models to simplify the analysis of the more complex processes of chromatin folding along the cell cycle, as revealed by differential polarization imaging and micro-spectropolarimetry.
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