Time-Resolved Confocal Fluorescence Microscope with Single Molecule Sensitivity

项目摘要 本申请寻求资金购买时间分辨共聚焦荧光显微镜， 分子灵敏度该仪器将支持NIH资助的三个部门和三个学院的用户 德克萨斯A&M大学社区内，并将提供大量的单分子荧光（SMF） 通过新的共享用户设施，十几个实验室目前无法获得这些能力。请求的五行 脉冲激光系统将使众多的多色相关，荧光寿命，和偏振 通过其时间相关单光子计数（TCSPC）功能， 组最常见的应用将是荧光共振能量转移（FRET）测量 用于确定大分子复合物中的分子内和分子间距离信息。 虽然整体FRET测量经常受到各种人为因素和限制的影响，但单个FRET测量通常不受影响。 所要求的显微镜系统的分子FRET（smFRET）能力大大提高了精确度 并提供了独特的方法来量化分子动力学。一个额外的关键应用 提出的仪器的发展将是一种新的方法来测量旋转流动性，称为 微秒时间尺度上的单分子旋转扩散显微镜（µs-SiMRoD）。旋转移动性 是一种未充分利用的实验读数，可用于探测大分子拥挤或其他 类似的情况下，分子组装影响探针迁移率。SiMRoD没有对应的 合奏模拟，强调仪器的单分子灵敏度的重要性。虽然 大多数用户将检查控制良好的体外系统，细胞FRET成像将受益于 荧光寿命显微镜（FLIM）系统的能力，这将产生更准确的 测量（FLIM-FRET）比典型的共聚焦显微镜更有效。主要用户将 研究基本的和不同的细胞生物学和机械生物化学问题，重点是 磷酸肌醇结合蛋白，核小体，分子伴侣，膜分裂，核孔，和 线粒体完整性用户组中代表的所有学院和部门将为以下内容做出贡献： 仪器成本，强调了新的显微镜功能在增长中的根本重要性 目前的研究计划。该仪器将被安置在医学院由系 分子和细胞医学，捐赠了大量的设备和空间的显微镜设施。 总的来说，已确定的用户计划了新的研究方向，这将需要超过93%的总数 可利用的用户时间，表明对现有项目和新项目的大量需求。总的来说， 具有单分子灵敏度的时间分辨共聚焦荧光显微镜将提供大量的 为广泛的项目提供基本的基础设施支持，对了解和改善 人体健康

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.