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Total Internal Reflection Fluorescence Microscope

全内反射荧光显微镜

基本信息

批准号：
7791846
负责人：
Gaudenz Danuser
金额：
$ 31.12万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-20 至 2011-05-19
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：我们请求资金购买全内反射荧光 (TIRF) 显微镜，该显微镜能够几乎同时对 3 和 4 个光谱不同的荧光团进行成像（颜色之间的切换时间 < 50 毫秒）。目前哈佛医学院校园内还没有这样的仪器。我们有五个主要用户。应用范围从单个分子马达的单分子追踪，到使用 FRET 和/或比例生物传感器对重建细胞骨架组件、细胞表面受体和细胞内信号转导进行分子动力学成像。七个小用户根据不同的需求添加了更多应用，证明购买灵活、模块化的最先进仪器是合理的。该显微镜将配备 7 条激光线。使用具有光纤出口孔径相对于物镜后焦平面的快速机动对准的 TIRF 照明器，可以准同时实现所有颜色的真正 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。光纤出口孔径的电动对准还可用于动态改变 TIRF 场的深度。我们建议为该仪器配备一个传统的小像素冷却CCD相机，适合对大分子组装体和生物传感器进行成像，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，但空间分辨率较粗糙。在为期一个月的 TIRF 显微镜与多光谱成像能力的并列比较中，PI 实验室对所提出的每一项功能和配件都进行了彻底评估。该仪器将由细胞生物学、系统生物学以及生物化学与分子药理学系共享显微镜成像设施的经验丰富的工作人员安装和管理。设施内已留出适当的空间。这种安排对于用户的持续培训和维护来说是最佳的，保证了该仪器投资的长期使用。