THE PHYSICAL CHEMISTRY OF NUCLEIC ACIDS

这次申遗是努力的延续，启动三年 在我们的实验室里，为了发展微分技术， 偏振显微镜及其在各种结构问题中的应用 具有生物医学意义。 在过去的三年里，我们已经建立了两个 差示偏振显微镜的不同原型。 第一 一种使用析像器照相机，第二种，使用共焦光学器件， 比第一个有了很大的进步 这些文书 已经过测试，并被用来证明这一力量， 复杂亚细胞结构的结构解析技术。 与这些发展并行，我们导出了测量理论， of this imaging成像technology技术. 所得表达式已用于 从所研究的系统中提取定量结构信息。 我们 我也开始使用这种方法来研究高阶 染色体的结构，并进行了实验和理论 与所述光学活性信号相关联的光学活性信号的表征 体外DNA缩合过程。 在本申请中，我们提出了一些研究，其最终目标是 为了阐明染色质沿着 细胞周期 为此，我们建议将联合收割机的光谱和 微分偏振显微镜的分析能力 最近发展的荧光显微镜技术。 微分 偏振成像将用于跟踪原位， 染色质沿着细胞周期的手性，并研究 中期染色体的高级结构。 荧光 显微镜技术将被用来跟踪，在实时，实际的 染色质和单个纤维的浓缩过程 DNA分子，在控制条件下。 这里提出的实验具有层次的复杂性。 在- 多聚赖氨酸诱导的单DNA分子凝聚的体外研究， 将被用作一个框架来分析和分析的结果， 研究旨在遵循，用同样的方法，盐诱导的 染色质在体外的凝聚。 最后，这些研究也将 作为模型，以简化分析更复杂的过程， 差异显示染色质沿着细胞周期折叠 偏振成像和显微偏振光谱法。

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.