TIRFM-imaging system for in vitro and in vivo cell biology

描述（由申请人提供）：本提案的目标是获得能够同时采集双色（绿色、红色）和几乎同时采集第三种颜色（远红）的全内反射（TIRF）显微镜。该显微镜还将配备光漂白/光转换能力。这一设备将被用作定量分析生物化学和活细胞动态过程的主要研究工具。生物科学系有五个主要用户（达特茅斯学院）和生物化学系（达特茅斯的Geisel医学院），其研究项目旨在了解细胞过程的分子机制，如肌动蛋白动力学、隔蛋白动力学、纤毛功能期间的鞭毛内转运、来自内质网出口位点的COPII转运、和一种新的细胞骨架元件（Pil 1细丝）的功能。在所有情况下，多个细胞成分都在亚秒的时间尺度上经历动态变化，包括细胞骨架和细胞膜。这些研究人员要实现和扩大资助研究的目标，就需要能够以高空间和时间分辨率以及单分子灵敏度监测多分子的动态。通过限制激发深度，TIRF显微镜已成为一种广泛使用的技术，以获得高的空间分辨率和增加的检测灵敏度。iXON相机和相关设备允许同时捕获两个荧光信号，这是亚秒级动态过程的必要条件。此外，第三个荧光信号可以在其他两个信号的0.2秒内检测到。EM-CCD相机允许在中等激光功率下的单分子灵敏度，最大限度地减少光漂白。这台仪器将是达特茅斯唯一的一台，并将为整个达特茅斯生命科学界服务。

DESCRIPTION (provided by applicant): The goal of this proposal is to acquire a total internal reflection (TIRF) microscope capable of simultaneous two-color acquisition (green, red) and near-simultaneous acquisition of a third color (far-red). The microscope would also be equipped with photobleaching/photoconversion capability. This equipment would be used as a primary research tool for quantitative analysis of dynamic processes, both biochemically and in live cells. There are five major users in the Department of Biological Sciences (Dartmouth College) and Department of Biochemistry (Geisel School of Medicine at Dartmouth) whose research projects are aimed at understanding molecular mechanisms of cellular processes such as actin dynamics, septin dynamics, intraflagellar transport during cilliary function, COPII transport from endoplasmic reticulum exit sites, and the function of a novel cytoskeletal element (Pil1 filaments). In all cases, multiple cellular elements are undergoing dynamics at sub-second timescales, including cytoskeleton and cellular membranes. Attainment and expansion of the goals of the funded research by these investigators requires the ability to monitor the dynamics of multiple molecules at high spatial and time resolution, and at single-molecule sensitivity. By limiting the excitation depth, TIRF microscopy has become a widely used technique to attain high spatial resolution and increased detection sensitivity. The iXON cameras and associated equipment allows simultaneous capture of two fluorescent signals, a necessity for processes with sub-second dynamics. In addition, a third fluorescent signal can be detected within 0.2 seconds of the other two signals. The EM-CCD camera allows single molecule sensitivity at moderate laser powers, minimizing photobleaching. This instrument would be the only one of its kind at Dartmouth and would serve the entire Dartmouth life science community.