IN VIVO FLUORESCENCE FLUCTUATION SPECTROSCOPY

体内荧光涨落光谱

• 批准号: 6701748
• 负责人: JOACHIM D MUELLER
• 金额: $ 14.44万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6701748
• 关键词: HeLa cells; bioengineering /biomedical engineering; bioimaging /biomedical imaging; biomedical equipment development; biophysics; cell biology; charge coupled device camera; chemical kinetics; fluorescence microscopy; fluorescence spectrometry; green fluorescent proteins; intermolecular interaction; intravital microscopy; method development; model design /development; molecular assembly /self assembly; nuclear receptors; optics; physical model; protein structure function; statistics /biometry; structural biology; time resolved data; transcription factor

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.

描述（由申请人提供）：荧光的独特特性 涨落光谱（FFS）使这项技术对细胞 应用. FFS不需要外部扰动来确定动力学和 具有亚微米空间分辨率和单分子的分子特性 灵敏度特别是，FFS在绿色标记蛋白质中的应用 荧光蛋白（GFP）具有彻底改变体内研究的潜力。 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 实现了体内研究的潜力。这项新技术的影响 将在许多生物学领域中感受到，其应用范围从基本的 从细胞生物学研究到药物筛选。 荧光相关光谱（FCS）使用自相关函数 以确定动力学参数，例如扩散系数。此外 常规FCS的另外两种涨落技术，光子计数直方图 (PCH)将使用扫描FCS。PCH是最近开发的技术， 决定了分子的亮度，这将被用来解决 蛋白质的结合。扫描FCS有可能检测固定的 proteins.总之，波动技术提供了互补的 成功表征生物化学物质所需信息 在体内的过程。为了评估这些技术在体内的真正潜力， 将进行彻底的表征， 每一项技术都将得到特别的重视。绿色荧光蛋白的性质及其在 将表征不同细胞区室中的自发荧光 为了了解FFS测量的定量范围， 细胞 波动技术将应用于维甲酸X受体（RXR）的研究， 一种核受体。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， 激素受体RXR的低聚状态，其范围从四聚体 单体，作为其激活的控制机制。的分辨率 利用涨落技术研究RXR的寡聚化状态将成为研究的重点 在这个体内研究中。此外，还将研究RXR与DNA的结合。