Development of Single-Biomolecule Optical Imaging

单生物分子光学成像的发展

• 批准号: 6748095
• 负责人: William E Moerner
• 金额: $ 22.7万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6748095
• 关键词: CHO cells; MHC class II antigen; T cell receptor; beta galactosidase; binding proteins; bioimaging /biomedical imaging; biomedical equipment development; biotechnology; cholesterol; enzyme activity; eukaryote; fluorescence resonance energy transfer; fluorescent dye /probe; green fluorescent proteins; magnetic field; major histocompatibility complex; membrane activity; method development; nanotechnology; optics; protein binding; protein folding; single cell analysis; spectrometry

DESCRIPTION (provided by applicant): The primary objectives of the proposed work are to develop improved methods of single-biomolecule optical detection and spectroscopy, and to apply these methods to several key biological problems of importance. The important advantage of the single-molecule regime is that signatures of conformational state and dynamical changes which are normally buried in ensemble experiments will become uniquely accessible to study. The development of techniques for single-biomolecule optical imaging and spectroscopy rests upon a balanced combination of (i) enhancement and extension of established techniques driven by specific biological applications, and (ii) exploratory investigation of new detection modalities. A new type of microscopy based on scanning of electromagnetically enhanced metal structures will be developed, which should lead to higher sensitivities as well as higher resolution. Specific biological problems of importance will be used to challenge the techniques, in order to stimulate the development of new capabilities. The principal biological problems of interest are: (a) detection and imaging of the diffusion of single copies of major histocompatibility complexes of type II in living cells in the presence of varying membrane cholesterol concentrations, binding proteins, and during T-cell receptor interactions, (b) measurements of the single-molecule enzymatic behavior of beta-galactosidase with an aim to explore the underlying stochastic dynamics in the absence or presence of various inhibitors, and (c) the observation and understanding of assisted protein folding in single bacterial and eukaryotic chaperones using a variety of reporter fluorophore technologies. To accomplish these ends, the Pl's physical, chemical, and optical expertise will be combined with the molecular biological, biochemical, and synthetic expertise of several collaborators. In the final analysis, owing to its multi-disciplinary organization, the fundamental research in this program will not only generate new knowledge about the biophysical properties of several important protein systems at the single-molecule level, but, in addition the advances in instrumentation will provide novel groundwork for technology transfer to other relevant disciplines.

描述（由申请人提供）：拟议工作的主要目的是开发改进的单双分子光学检测和光谱的方法，并将这些方法应用于重要的几个关键生物学问题。单分子制度的重要优点是，构象状态和通常埋葬在整体实验中的动态变化的特征将变得独特地进行研究。单生物分子光学成像和光谱法的技术的发展取决于（i）由特定生物学应用驱动的已建立技术增强和扩展的平衡组合，以及（ii）对新检测方式的探索性研究。将开发基于电磁增强金属结构的扫描的新型显微镜，这将导致更高的敏感性以及更高的分辨率。为了刺激新能力的发展，将使用特定的重要生物学问题来挑战这些技术。 The principal biological problems of interest are: (a) detection and imaging of the diffusion of single copies of major histocompatibility complexes of type II in living cells in the presence of varying membrane cholesterol concentrations, binding proteins, and during T-cell receptor interactions, (b) measurements of the single-molecule enzymatic behavior of beta-galactosidase with an aim to explore the underlying stochastic dynamics in使用各种报告基因的荧光团技术，对单个细菌和真核生物伴侣的辅助蛋白折叠的观察和理解（c）不存在或存在各种抑制剂。 为了实现这些目的，PL的物理，化学和光学专业知识将与几个合作者的分子生物学，生化和合成专业知识相结合。在最终分析中，由于其多学科组织，该计划中的基本研究不仅将在单分子水平上产生有关几种重要蛋白质系统的生物物理特性的新知识，而且，仪器的进步还将为技术转移到其他相关学科的技术转移提供新的基础。