In Vivo fluorescence correlation spectroscopy

描述（由申请人提供）：拟议研究的目的是 将荧光相关光谱学发展成为一种有效、可靠的工具 用于动态过程的体内表征。这项技术具有巨大的 生物医学应用的潜力，如体内药物筛选。 荧光相关光谱法（FCS）研究荧光 激光焦点内少量分子的波动 使用统计分析工具。在FCS提供的独特信息中 是绝对荧光团浓度和动力学参数，例如 扩散系数和化学反应系数。FCS提供动态 信息与亚微米空间分辨率，而无需应用 外部扰动使其特别吸引人， 测量.另一种最近开发的基于光子的分析技术 将采用计数直方图来提取有关 FCS数据的亮度不均匀性。一个仔细的实验检查， 将进行绿色荧光蛋白（GFP）的体内特性， 同时明确考虑蜂窝环境对FCS的影响 测量.此外，GFP标记的类维生素A受体将用于 进行体内研究，以解析它们的分子寡聚化状态， 配体的存在和不存在。

DESCRIPTION (provided by applicant): The purpose of the proposed research is to develop fluorescence correlation spectroscopy into an effective, reliable tool for in vivo characterization of dynamic processes. This technology has enormous potential for biomedical applications, such as in vivo drug screening. Fluorescence correlation spectroscopy (FCS) studies the fluorescence fluctuations of a small number of molecules inside the focal spot of a laser with statistical analysis tools. Among the unique information provided by FCS is the absolute fluorophore concentration and kinetic parameters, such as the diffusion coefficient and chemical reaction coefficients. FCS delivers dynamic information with submicron spatial resolution and without the need to apply an external perturbation rendering it especially attractive for in vivo measurements. Another recently developed analysis technique based on the photon counting histogram will be employed to extract additional information on brightness heterogeneity from FCS data. A careful experimental examination of the in vivo properties of green fluorescent protein (GFP) will be conducted, while explicitly considering the influence of the cellular environment on FCS measurements. In addition, GFP tagged retinoid receptors will be used to perform an in vivo study to resolve their molecular oligomerization state in the presence and absence of ligands.