Total Internal Reflection Fluorescence Microscope

描述(由申请人提供)：我们申请资金购买全内反射荧光(TIRF)显微镜，能够几乎同时成像3和4个光谱不同的荧光团(&lt；50ms颜色切换时间)。哈佛医学院的校园里目前还没有这样的仪器。我们有五个主要用户。应用范围从单个分子马达的单分子跟踪到重组细胞骨架组件、细胞表面受体的分子动力学成像，以及使用FRET和/或比率生物传感器的细胞内信号转导。七个较小的用户增加了更多具有不同需求的应用程序，证明了购买灵活和模块化的最先进仪器是合理的。显微镜将配备7条激光线路。使用TIRF照明器，光纤出射光圈相对于物镜背焦面快速电动对准，可以准同时实现所有颜色的真实TIRF照明。在这种情况下，特定实验的颜色组合仅受可用二向色镜的多带通特性的限制。还可以利用光纤出射孔径的电动对准来动态改变TIRF场的深度。我们建议为该仪器配备一个小像素尺寸的传统制冷CCD相机，适合于成像大分子组件和生物传感器，并配备一个电子倍增CCD相机，为单分子检测提供精致的灵敏度，但空间分辨率较差。在对具有多光谱成像能力的TIRF显微镜进行了长达一个月的并排比较期间，PI的实验室对每个建议的功能和附件进行了彻底的评估。该仪器将由细胞生物学、系统生物学和生物化学与分子药理学系的共享显微镜成像设施的经验丰富的工作人员安装和管理。设施中已预留了适当的空间。这一安排对于用户的持续培训和维护将是最佳的，从而保证长期使用这一仪器的投资。

DESCRIPTION (provided by applicant): We request funds to purchase a Total Internal Reflection Fluorescence (TIRF) microscope with the capability for imaging 3 and 4 spectrally distinct fluorophores near-simultaneously (< 50ms switching time between colors). No such instrument is currently available on the campus of the Harvard Medical School. We have five major users. Applications range from single molecule tracking of individual molecular motors to imaging of molecular dynamics of reconstituted cytoskeleton assemblies, cell surface receptors, and intracellular signal transduction using FRET and/or ratiometric biosensors. Seven minor users add further applications with diverse needs, justifying the acquisition of a flexible and modular state-of-the-art instrument. The microscope will be equipped with 7 laser lines. Using a TIRF-illuminator with fast motorized alignment of the fiber exit aperture relative to the objective back focal plane, true TIRF illumination can be achieved for all colors quasi-simultaneously. The color combinations for a particular experiment are in this case restricted only by the multi-bandpass characteristic of available dichroic mirrors. The motorized alignment of the fiber exit aperture can also be exploited to alter the depth of the TIRF field on the fly. We propose to equip the instrument with a conventional cooled CCD camera with small pixel size, appropriate for imaging macromolecular assemblies and biosensors, and with an electron-multiplying CCD camera, providing exquisite sensitivity for single molecule detection but coarser spatial resolution. Each of these proposed features and accessories has been thoroughly evaluated in the lab of the PI during a month long side-by-side comparison of TIRF microscopes with multi-spectral imaging capacity. The instrument will be installed and administered by highly-experienced staff of the shared microscope imaging facility of the Departments of Cell Biology, Systems Biology, and Biochemistry & Molecular Pharmacology. Appropriate space in the facility has been set aside. This arrangement will be optimal for the continued training of users and maintenance, warranting long-term use of this instrument investment.