IN VIVO FLUORESCENCE FLUCTUATION SPECTROSCOPY

描述(申请人提供)：荧光的独特特征 涨落光谱(FFS)使这项技术对细胞有吸引力 申请。FFS不需要外部摄动来确定动力学和 具有亚微米空间分辨率的单分子分子性质 敏感度。特别是FFS在绿色标记蛋白质中的应用 荧光蛋白(GFP)有可能给体内研究带来革命性的变化。 拟议研究的长期目标在于同时 波动技术的发展和应用，使其充分发挥作用 体内研究的潜力已经实现。这项新技术的影响 将在许多生物领域感受到，应用范围从基本的 细胞生物学研究在药物筛选中的应用。 荧光相关光谱(FCS)使用自相关函数 以确定动力学参数，如扩散系数。此外 为了规范FCS的另外两种起伏技术，光子计数直方图 (PCH)和扫描FCS。PCH，一种最近发展起来的技术， 确定分子亮度，该亮度将用于解决 蛋白质的结合。扫描FCS有可能检测到固定化 蛋白质。总之，波动技术提供了补充 成功描述生化特性所需的信息 活体内的过程。为了评估这些技术在体内的真正潜力 将进行彻底的表征，并将统计准确性 每项技术都将得到特别重视。研究了绿色荧光蛋白的性质以及绿色荧光蛋白 不同细胞隔间的自体荧光将被表征 为了了解生活中FFS测量的量化范围 细胞。 涨落技术将用于研究维甲酸X受体(RXR)， 一种核受体，在体内。核受体受配体控制 转录调节因子和RXR已被确定为关键调节因子 荷尔蒙受体。RXR的齐聚状态，范围为四聚体 对于单体，作为其激活的控制机制。的决议案 涨落技术对RXR的寡化状态将是焦点 这项活体研究的结果。此外，还将研究RXR与DNA的结合。

DESCRIPTION (provided by applicant): The unique features of fluorescence fluctuation spectroscopy (FFS) make this technique attractive for cellular applications. FFS requires no external perturbation to determine kinetic and molecular properties with submicron spatial resolution and single molecule sensitivity. Especially, the application of FFS to proteins tagged with green fluorescent protein (GFP) has the potential to revolutionize in vivo studies. The long-term objective of the proposed research lies in the concurrent development and application of fluctuation techniques, so that their full potential for in vivo studies is realized. The impact of this new technology will be felt in many biological areas with applications ranging from basic research in cell biology to pharmaceutical drug screening. Fluorescence correlation spectroscopy (FCS) uses the autocorrelation function to determine kinetic parameters, such as the diffusion coefficient. In addition to regular FCS two other fluctuation techniques, the photon counting histogram (PCH) and scanning FCS will be used. PCH, a recently developed technique, determines the molecular brightness, which will be used to address the association of proteins. Scanning FCS has the potential to detect immobilized proteins. Together, the fluctuation techniques provide complementary information necessary for a successful characterization of biochemical processes in vivo. To assess the true potential of these techniques in vivo a thorough characterization will be performed and the statistical accuracy of each technique will receive special emphasis. The properties of EGFP and the autofluorescence in the different cellular compartments will be characterized in order to understand the quantitative range of FFS measurements in living cells. The fluctuation techniques will be applied to study Retinoid X Receptor (RXR), a nuclear receptor, in vivo. Nuclear receptors are ligand controlled transcription regulators and RXR has been identified as the key regulator of hormone receptors. The oligomerization state of RXR, which ranges from tetramer to monomer, serves as a control mechanism for its activation. The resolution of the oligomenzation state of RXR by fluctuation techniques will be at the focus of this in vivo study. In addition, the binding of RXR to DNA will be studied.