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

用于体外和体内细胞生物学的 TIRFM 成像系统

• 批准号: 8639757
• 负责人: Amy Susanne Gladfelter
• 金额: $ 34.86万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8639757
• 关键词: Actins; Beryllium; Biochemistry; Biological Sciences; Cell physiology; Cells; Cellular Membrane; Cellular biology; Color; Communities; Cytoskeleton; Detection; Elements; Endoplasmic Reticulum; Equipment; Filament; Funding; Goals; Image; In Vitro; Lasers; Life; Microscope; Microscopy; Molecular; Monitor; Photobleaching; Process; Research; Research Personnel; Research Project Grants; Resolution; Signal Transduction; Site; System; Techniques; Time; charge coupled device camera; college; in vivo; instrument; medical schools; novel; single molecule; tool

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.

描述（由申请人提供）：本提案的目标是获得一种全内反射（TIRF）显微镜，能够同时采集两种颜色（绿色、红色）和近乎同时采集第三种颜色（远红色）。该显微镜还将配备光漂白/光转换功能。该设备将用作生物化学和活细胞动态过程定量分析的主要研究工具。生物科学系（达特茅斯学院）和生物化学系（达特茅斯盖泽尔医学院）有五个主要用户，他们的研究项目旨在了解细胞过程的分子机制，例如肌动蛋白动力学、隔膜动力学、纤毛功能期间的鞭毛内运输、内质网出口位点的 COPII 运输以及新型细胞骨架元件的功能 （Pil1 细丝）。在所有情况下，多个细胞元件都在亚秒级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展资助研究的目标，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个分子的动力学。通过限制激发深度，TIRF 显微镜已成为一种广泛使用的技术，可实现高空间分辨率和提高检测灵敏度。 iXON 相机和相关设备可以同时捕获两个荧光信号，这是亚秒级动态过程所必需的。此外，第三个荧光信号可以在其他两个信号的 0.2 秒内被检测到。 EM-CCD 相机可在中等激光功率下实现单分子灵敏度，从而最大限度地减少光漂白。该仪器将是达特茅斯唯一的同类仪器，将为整个达特茅斯生命科学界服务。

项目成果

SABRE populates ER domains essential for cell plate maturation and cell expansion influencing cell and tissue patterning.

• DOI: 10.7554/elife.65166
• 发表时间: 2021-03-09
• 期刊: eLife
• 影响因子: 7.7
• 作者: Cheng X;Bezanilla M
• 通讯作者: Bezanilla M

In vivo analysis of formin dynamics in the moss P. patens reveals functional class diversification

• DOI: 10.1242/jcs.233791
• 发表时间: 2020-01
• 期刊: Journal of Cell Science
• 影响因子: 4
• 作者: Peter A C van Gisbergen;Shu-Zon Wu;Xiaohang Cheng;Kelli A. Pattavina;M. Bezanilla