High Speed Confocal Photomanipulation Microscopyfor use in multi-user facility

项目概述：蛋白质的功能取决于蛋白质在细胞环境中与底物或伴侣蛋白相互作用的可用性。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，可以直接定量活细胞中的蛋白质可用性。选择性光漂白或光活化（光电操纵）的群体荧光标记的蛋白质允许研究人员测量蛋白质的流动性，分子大小，移动蛋白的百分比，并遵循光活化蛋白的命运。虽然荧光成像和光电操纵为蛋白质动力学提供了许多见解，但这种方法的一个严重缺陷是数据采集速度相对较慢（每张图像的十分之一秒），以及光电操纵事件结束和收集第一张操作后图像之间的延迟。这对细胞质或管状蛋白来说尤其成问题，因为它们可以在几毫秒的时间内移动几微米。蔡司的一种新型共聚焦光电操纵显微镜，Duo，以其卓越的图像质量、量子效率和速度打破了这一时间障碍。这里提出的蔡司双星系统将是一个多用户设施的一部分，不能只由一个实验室使用。此应用程序的目的是为AECOM社区提供目前无法获得的技术。目前校园里的设备都不适合同时进行高速共聚焦成像，同时进行快速感兴趣区域光漂白或活材料的光活化，用于蛋白质迁移率测量。该应用程序主要用户的共同科学需求是能够从活细胞中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，我们要求的系统将为专家用户组解决速度、灵敏度和定量问题。相关性：细胞蛋白的错误调控发生在从癌症到阿尔茨海默病的疾病中。为了确定细胞蛋白的正常和疾病特性，确定细胞蛋白的环境、结合伙伴和动力学是至关重要的。本提案中所描述的高速显微镜将满足我们研究小组的这一需求。

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.