High Speed Confocal Photomanipulation Microscopyfor use in multi-user facility

用于多用户设施的高速共焦光学显微镜

• 批准号: 7209582
• 负责人: Robert H Singer
• 金额: $ 39.73万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7209582
• 关键词: Alzheimer' s Disease; Binding; Budgets; Cells; Collection; Communities; Coupled; Disease; Environment; Equipment; Event; Green Fluorescent Proteins; Image; Label; Laboratories; Laser Scanning Confocal Microscopy; Life; Malignant Neoplasms; Measurement; Measures; Methodology; Microscope; Microscopy; Photobleaching; Population; Problem Solving; Property; Protein Dynamics; Proteins; Purpose; Range; Rate; Research; Research Personnel; Resolution; Speed; System; Technology; cell growth regulation; data acquisition; fluorescence imaging; insight; interest; millisecond; molecular size; photoactivation; protein function; quantum

DESCRIPTION (provided by applicant): High Speed Confocal Photomanipulation Microscopy for use in multi-user facility Project Summary Protein function depends on the availability of a protein to interact with substrates or partner proteins in the cellular environment. By labeling proteins with green fluorescent protein and applying laser scanning confocal microscopy, it has become possible to directly quantitate protein availability in live cells. Selective photobleaching or photoactivation (photomanipulation) of a population of fluorescently labeled proteins permits investigators to measure a protein's mobility, molecular size, percent of mobile proteins, and follow a photoactivated protein's fate. While fluorescence imaging and photomanipulation have provided many insights into protein dynamics, a serious deficit in this methodology has been the relatively slow rates of data acquisition (tenths of seconds per image) and the delay between the end of a photomanipulation event and the collection of the first postmanipulation images. This is especially problematic for cytoplasmic or lumenal proteins that can move several microns in the space of milliseconds. A new confocal photomanipulation microscope from Zeiss, the Duo, has broken this temporal barrier with remarkable image quality, quantum efficiency, and speed. The Zeiss Duo system proposed here will be part of a multi-user facility and cannot be used exclusively by one laboratory. The purpose of this application is to provide technology to the AECOM community that is not currently available. None of the equipment currently on campus is suited to the simultaneous high speed confocal imaging coupled with rapid region of interest photobleaching or photoactivation of live material for protein mobility measurements. The common scientific need of the major users in this application is the ability to obtain high spatial and temporal resolution images from live cells for quantitative protein mobility measurements. As discussed in the budget justification, the system we request will solve the problems of speed, sensitivity, and quantitation for an expert user group. Relevance: Mis-regulation of cellular proteins occurs in diseases ranging from cancer to Alzheimer's disease. To define the normal and disease properties of cellular proteins, it is critical to identify the environments, binding partners, and dynamics of cellular proteins. The high speed microscope described in this proposal will fulfill this need for our research groups.

描述（由申请人提供）：用于多用户设施的高速共聚焦光操纵显微镜项目摘要蛋白质功能取决于蛋白质与细胞环境中的底物或伴侣蛋白质相互作用的可用性。通过用绿色荧光蛋白标记蛋白质并应用激光扫描共聚焦显微镜，已经可以直接定量活细胞中的蛋白质可用性。荧光标记蛋白质群体的选择性光漂白或光活化（光操纵）允许研究人员测量蛋白质的迁移率、分子大小、移动的蛋白质的百分比，并跟踪光活化蛋白质的命运。虽然荧光成像和photomanipulation提供了许多见解蛋白质动力学，在这种方法中的一个严重缺陷是相对缓慢的数据采集速率（每幅图像的十分之一秒）和延迟之间的photomanipulation事件结束和收集的第一postmanipulation图像。这对于可以在毫秒空间内移动几微米的细胞质或内腔蛋白质来说尤其成问题。蔡司的一种新的共焦光操纵显微镜，Duo，以卓越的图像质量，量子效率和速度打破了这一时间障碍。这里提出的蔡司Duo系统将是多用户设施的一部分，不能仅由一个实验室使用。此应用程序的目的是为AECOM社区提供目前不可用的技术。目前校园里没有一台设备适合同时进行高速共聚焦成像，并结合快速感兴趣区域的光漂白或活物质的光活化，用于蛋白质迁移率测量。该应用中主要用户的共同科学需求是能够从活细胞获得高空间和时间分辨率图像，用于定量蛋白质迁移率测量。正如预算论证中所讨论的，我们要求的系统将解决专家用户组的速度、灵敏度和定量问题。相关性：从癌症到阿尔茨海默病，细胞蛋白质的错误调节发生在各种疾病中。为了确定细胞蛋白质的正常和疾病特性，识别细胞蛋白质的环境、结合伴侣和动力学是至关重要的。本提案中描述的高速显微镜将满足我们研究小组的这一需求。