Time-Resolved Confocal Fluorescence Microscope with Single Molecule Sensitivity

项目总结 这项申请寻求资金购买时间分辨共聚焦荧光显微镜与单一 分子敏感性。该仪器将支持美国国立卫生研究院资助的三个系和三所学院的用户 在德克萨斯农工大学社区内，并将提供大量单分子荧光(SMF) 目前十几个实验室无法通过一个新的共享用户设施获得这些功能。请求的五行代码 脉冲激光系统将实现许多多色关联、荧光寿命和偏振 通过用户所需的时间相关单光子计数(TCSPC)功能 一群人。最常见的应用将是荧光共振能量转移(FRET)测量 用于确定大范围大分子络合物中的分子内和分子间距离信息。 虽然整体FRET测量经常受到各种伪像和限制的影响，但单一的 所要求的显微镜系统的分子FRET(SmFRET)能力大大提高了精度 并提供了量化分子动力学的独特方法。一个额外的关键应用程序 将开发一种测量旋转迁移率的新方法，称为 微秒时间尺度上的单分子旋转扩散显微镜(µS-SiMRoD)。旋转机动性 未充分利用的实验读数对于探测大分子拥挤或其他因素的影响有用吗 分子组装影响探针迁移率的类似情况。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 大多数用户将在体外检查控制良好的系统，在纤维素酶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 与典型的共焦显微镜相比，它的测量结果(Flim-FRET)更好。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 磷脂酰肌醇结合蛋白，核小体，分子伴侣，膜分裂，核孔，和 线粒体的完整性。用户组中代表的所有学院和院系都将为 仪器成本，强调新显微镜能力在增长中的根本重要性 目前的研究项目。该仪器将存放在医学院，由 分子和细胞医学，它已经为显微镜设施捐赠了大量的设备和空间。 总体而言，已确定的用户计划了新的研究方向，这将需要总数的93%以上 可获得的用户时间，表明对现有项目和新项目的大量需求。总体而言，请求的 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和 为广泛的项目提供基础设施支持，这些项目对理解和改进很重要 人类健康。

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.