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）：该提案的目的是获取能够同时进行两色（绿色，红色）和近距离获取第三种颜色（Far-Red）的总内部反射（TIRF）显微镜。显微镜还将配备光漂白/光转换能力。该设备将用作生化和活细胞中动态过程进行定量分析的主要研究工具。 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细丝）。在所有情况下，多个细胞元素都在次秒时间尺度（包括细胞骨架和细胞膜）进行动力学。这些研究者资助研究目标的实现和扩展需要能够在高空间和时间分辨率以及单分子敏感性下监测多个分子的动态。通过限制激发深度，TIRF显微镜已成为一种广泛使用的技术，可实现高空间分辨率并提高检测灵敏度。 IXON摄像机和相关的设备允许同时捕获两个荧光信号，这是具有下秒动力​​学的过程的必要性。另外，可以在其他两个信号的0.2秒内检测到第三个荧光信号。 EM-CCD摄像机允许在中等激光功率下进行单分子敏感性，从而最大程度地减少光漂白。该乐器将是达特茅斯（Dartmouth）唯一同类乐器，它将为整个达特茅斯生活科学界提供服务。

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