Protein Translational Mobility and Concentration Near Membrane Surfaces

将使用具有荧光相关光谱的全内反射来确定控制沉积在透明平面基底上的单个磷脂双层附近的荧光蛋白的平移迁移率和浓度的因素。 氩离子激光束将在基材/溶液界面处发生内反射，产生仅延伸到相邻溶液中一小段距离的倏逝场。 沿着渐逝深度，放置在光学显微镜的中间像平面处的孔径将限定紧挨着膜表面的小体积。 由这个小体积内的分子产生的荧光，用高孔径物镜收集并用快速硅雪崩光电二极管检测，将随着单个荧光蛋白移入和移出观察体积而随时间波动。 标准化荧光波动自相关函数将提供有关溶质平移迁移率和浓度的信息。 许多生物过程涉及溶液中的分子（例如蛋白质）与生物细胞表面上存在的受体分子的相互作用。 这些相互作用的细节，导致特定的生物学行为，预测强烈依赖于直接邻近细胞表面的溶液中的蛋白质的浓度和运动。 这两个属性，浓度和平移迁移率，将测量荧光蛋白旁边的透明平面上构建的细胞表面模型，通过使用新开发的方法，采用激光激发和光学显微镜。 这些结果将用于扩展目前关于细胞如何检测和响应其环境的一般知识水平，从而产生生物功能。

9728116 Thompson Total internal reflection with fluorescence correlation spectroscopy will be used to determine the factors which govern the translational mobilities and concentrations of fluorescent proteins adjacent to single phospholipid bilayers deposited on transparent planar substrates. An argon ion laser beam will be internally reflected at the substrate/solution interface, creating an evanescent field that extends only a small distance into the adjacent solution. Along with the evanescent depth, an aperture placed at an intermediate image plane of an optical microscope will define a small volume next to the membrane surface. The fluorescence arising from molecules within this small volume, collected with a high aperture objective and detected with a fast silicon avalanche photodiode, will fluctuate with time as individual fluorescent proteins move in and out of the observed volume. The normalized fluorescence fluctuation autocorrelation function will provide information about solute translational mobility and concentration. A large variety of biological processes involve the interactions of molecules in solution, such as proteins, with receptor molecules present on the surfaces of biological cells. The details of these interactions, which result in specific biological behavior, are predicted to depend strongly on the concentrations and movements of the proteins in solution directly adjacent to the cell surfaces. These two properties, concentration and translational mobility, will be measured for fluorescent proteins next to models of cell surfaces constructed on transparent planar surfaces, by using newly developed methods that employ laser excitation and optical microscopy. The results will be used to expand the current level of general knowledge about how cells detect and respond to their environments, giving rise to biological function.