Time-Resolved Confocal Fluorescence Microscope with Single Molecule Sensitivity

具有单分子灵敏度的时间分辨共焦荧光显微镜

• 批准号: 10415601
• 负责人: SIEGFRIED M MUSSER
• 金额: $ 60万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10415601
• 关键词: Binding Proteins; Biochemistry; Biological; Cells; Color; Communities; Crowding; Development; Diffusion; Equipment; Fluorescence; Fluorescence Resonance Energy Transfer; Funding; Growth; Health; Human; Image; In Vitro; Infrastructure; Laboratories; Lasers; Macromolecular Complexes; Measurement; Measures; Medicine; Membrane; Microscope; Microscopy; Mitochondria; Molecular; Molecular Chaperones; Morphologic artifacts; Nuclear Pore; Nucleosomes; Phosphatidylinositols; Photons; Physiologic pulse; Research; Rotation; System; Texas; Time; United States National Institutes of Health; Universities; analog; college; cost; fluorescence microscope; improved; instrument; instrumentation; molecular assembly/self assembly; molecular dynamics; novel strategies; programs; single molecule; single-molecule FRET

PROJECT SUMMARY This application seeks funds to purchase a time-resolved confocal fluorescence microscope with single molecule sensitivity. This instrument will support NIH-funded users in three departments and three colleges within the Texas A&M University community and will provide numerous single molecule fluorescence (SMF) capabilities currently unavailable to a dozen laboratories via a new shared user facility. The requested five-line pulsed laser system will enable numerous multi-color correlation, fluorescence lifetime, and polarization applications through its time-correlated single photon counting (TCSPC) capabilities that are required by the user group. The most common application will be fluorescence resonance energy transfer (FRET) measurements used to determine intra- and inter-molecular distance information in a wide-range of macromolecular complexes. While ensemble FRET measurements are often compromised by various artifacts and limitations, the single molecule FRET (smFRET) capabilities of the requested microscope system substantially improve the accuracy of such measurements and provide unique ways to quantify molecular dynamics. An additional key application of the proposed instrument will be the development of a novel approach for measuring rotational mobility termed single molecule rotational diffusion microscopy on the microsecond timescale (µs-SiMRoD). Rotational mobility is an underutilized experimental read-out useful for probing the effects of macromolecular crowding, or other similar situations where the molecular assembly influences probe mobility. SiMRoD has no corresponding ensemble analog, emphasizing the importance of the instrument’s single molecule sensitivity. Though the majority of users will examine well-controlled in vitro systems, in cellulo FRET imaging will benefit from the fluorescence lifetime microscopy (FLIM) capability of the system, which will generate more accurate measurements (FLIM-FRET) than are available from a typical confocal microscope. The Major Users will examine fundamental and diverse cell biological and mechanistic biochemistry questions focused on phosphoinositide binding proteins, nucleosomes, molecular chaperones, membrane fission, nuclear pores, and mitochondrial integrity. All the colleges and departments represented in the user group will contribute to instrumentation costs, emphasizing the fundamental importance of the new microscope capabilities in the growth of current research programs. The instrument will be housed in the College of Medicine by the Department of Molecular and Cellular Medicine, which has donated substantial equipment and space for the microscope facility. Altogether, the identified users have planned new research directions that will require over 93% of the total accessible user time, indicating the substantial demand for both existing and new projects. In total, the requested time-resolved confocal fluorescence microscope with single molecule sensitivity will provide substantial and fundamental infrastructural support for a wide range of projects important for understanding and improving human health.

项目摘要 该应用程序寻求资金购买单一的时间分辨共聚焦荧光显微镜 分子灵敏度。该工具将支持三个系和三所大学的NIH资助的用户 在德克萨斯A＆M大学社区中，将提供大量的单分子荧光（SMF） 目前，通过新的共享用户设施无法使用十几个实验室的功能。请求的五线 脉冲激光系统将实现许多多色相关性，荧光寿命和极化 通过其时间相关的单个光子计数（TCSPC）功能的应用程序所需的功能 团体。最常见的应用是荧光共振能量传递（FRET）测量 用于确定大分子大分子复合物中的分子内和分子间距离信息。 虽然整体测量经常被各种工件和局限性损害，但单个 所需显微镜系统的分子颗粒（SMFRET）功能显着提高了准确性 这种测量值，并提供了量化分子动力学的独特方法。附加的关键应用程序 所提出的仪器将开发一种新型方法，用于测量所谓的旋转迁移率 微秒时间尺度（µS-SIMROD）上的单分子旋转扩散显微镜。旋转活动性 是一种未充分利用的实验读数，可用于探测大分子拥挤的影响或其他 分子组装影响探针迁移率的类似情况。 Simrod没有相应的 合奏类似物，强调仪器单分子灵敏度的重要性。虽然 大多数用户将检查体外系统控制良好的体外系统，在纤维素FRET成像中将受益于 系统的荧光寿命显微镜（FLIM）能力，这将产生更准确的 测量值（FLIM-FRET）比典型的共聚焦显微镜可用。主要用户将 研究基本和多样化的细胞生物学和机械生物化学问题的重点 磷酸肌醇结合蛋白，核小体，分子伴侣，膜裂变，核孔和 线粒体完整性。用户组中代表的所有学院和部门都将为 仪器成本，强调新显微镜能力在增长中的基本重要性 当前的研究计划。该仪器将由科 分子和细胞医学，已为显微镜设施捐赠了大量设备和空间。 总体而言，确定的用户计划了新的研究指示，这将需要超过93％的总数 可访问的用户时间，表明对现有项目和新项目的大量需求。总共请求 具有单分子敏感性的时间分辨共聚焦荧光显微镜将提供大量，并且 基础设施的基本基础设施支持广泛的项目，对于理解和改进 人类健康。