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）：荧光的独特特征 波动光谱（FFS）使该技术对细胞有吸引力 申请。 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，将FFS应用于用绿色标记的蛋白质 荧光蛋白（GFP）有可能在体内研究中彻底改变。 拟议研究的长期目标在于并发 波动技术的开发和应用，以使其充分 实现了体内研究的潜力。这项新技术的影响 在许多生物领域都会感受到，其应用范围从基本 细胞生物学研究对药物筛查的研究。 荧光相关光谱（FCS）使用自相关函数 确定动力学参数，例如扩散系数。此外 对于常规FCS，另外两种波动技术，光子计数直方图 （PCH）和扫描FC将使用。 PCH，最近开发的技术， 确定分子亮度，该亮度将用于解决 蛋白质的关联。扫描FCS有可能检测到固定的 蛋白质。波动技术共同提供了互补的 成功表征生化所需的信息 体内过程。评估这些技术在体内的真正潜力 将进行详尽的表征，并具有统计准确性 每种技术都会特别重视。 EGFP和 在不同的蜂窝室中的自动荧光将被表征 为了了解生活中FFS测量的定量范围 细胞。 波动技术将应用于研究类视视视视认为X受体（RXR）， 核受体，体内。核受体是配体控制的 转录调节器和RXR已被确定为关键调节剂 激素受体。 RXR的寡聚状态，范围从四聚体 对单体来说，是其激活的控制机制。解决方案 通过波动技术的RXR的寡聚状态将成为重点 这项体内研究。另外，将研究RXR与DNA的结合。