High Speed Confocal Photomanipulation Microscopyfor use in multi-user facility

描述(由申请人提供)：用于多用户设备的高速共聚焦光学操纵显微镜项目摘要蛋白质的功能取决于蛋白质在细胞环境中与底物或伙伴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和应用激光扫描共聚焦显微镜，可以直接定量活细胞中蛋白质的可获得性。选择性光漂白或对一组荧光标记蛋白质的光激活(光操纵)允许研究人员测量蛋白质的流动性、分子大小、可移动蛋白质的百分比，并跟踪光激活蛋白质的命运。虽然荧光成像和光操纵提供了对蛋白质动力学的许多见解，但这种方法的一个严重缺陷是数据采集速度相对较慢(每幅图像只有十分之一秒)，以及光操纵事件结束和第一批操作后图像收集之间的延迟。这对于可以在毫秒内移动几个微米的细胞质或管腔蛋白来说尤其成问题。蔡司的一种新的共焦光电操纵显微镜，这两个人，以惊人的图像质量、量子效率和速度打破了这一时间障碍。这里提出的蔡司双人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。该应用程序的目的是向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于同时进行高速共聚焦成像，并结合感兴趣区域的快速光漂白或活材料的光激活来测量蛋白质的迁移率。在这一应用中，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的图像，用于定量测量蛋白质的迁移率。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、敏感度和量化问题。相关性：从癌症到阿尔茨海默氏症等疾病中都会发生细胞蛋白的错误调节。要确定细胞蛋白质的正常和疾病性质，关键是要确定细胞蛋白质的环境、结合伙伴和动力学。这项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。

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.