INTRAVITAL VIDEO-RATE CONFOCAL MICROSCOPE

本申请申请的是一个视频速率共聚焦显微镜设备，用于活体活体显微镜。动物，如转基因小鼠，在流动室中滚动的活细胞和在组织培养中维持的活组织。我们希望结合三种先进技术来发展我们对血管疾病、皮肤病、血栓形成、细胞信号和细胞的理解：(1)活体小鼠血管显微术，研究血管系统的各种扰动对内皮细胞、白细胞、血小板和血浆蛋白（包括凝血蛋白）的影响；(2)通过基因敲除策略改变或转基因的小鼠的产生，其净效果是产生蛋白质缺乏或在感兴趣的蛋白质中引入荧光团；(3)开发视频速度共聚焦显微镜，如BioRad RTS2000和Noran OZ共聚焦扫描激光显微镜，可以实现快速、视频速率、高分辨率的实时数据采集。哈佛医学院的一个研究人员联盟已经成立，他们对视频速率共聚焦显微镜设备有特殊的需求。这个小组由一群初级和成熟的研究者组成，他们对视频速率共聚焦显微镜有特殊的需求，因为它可以应用于他们的nih资助的研究项目。目前在哈佛医学院或其附属医院没有这样的仪器。视频速率共聚焦显微镜系统必须包括以下关键特征：(1)用于活体动物的活体显微镜设备；(2)一种快速扫描、适用于流动条件下DIC和荧光显微镜的激光共聚焦显微镜系统；(3)装有成像软件的计算机工作站，用于图像采集和重建；(4)多荧光通道共定位实验。BioRad RTS2000实时激光共聚焦系统，安装在尼康E600FN显微镜上，将为一组研究人员提供仪器，以显着推进他们的领域。

This application is submitted for a video-rate confocal microscopy facility for intravital microscopy in living small. animals, such as genetically altered mice, in living cells rolling in a flow chamber and viable tissue maintained in tissue culture. We wish to merge three advanced technologies to develop our understanding of vascular disease, skin disease, thrombosi, cell signalling and cell: (1) intravital microscopy of blood vessels in living mice to study the impact of various perturbations of the vascular system on endothelium, leukocytes, platelets and plasma proteins including the blood clotting proteins; (2) the generation of mice altered by the knockout strategy or rendered transgenic, with the net effect of creating a protein deficiency or introducing a fluorophore into a protein of interest; (3) the development of video speed confocal microscopes, such as the BioRad RTS2000 and Noran OZ confocal scanning laser microscopes, that permit rapid, video rate, real-time data acquisition at high resolution. A consortium of investigators at Harvard Medical School has been established that have a special need for a video rate confocal microscope facility. This group is composed of a group of junior and established investigators who have a special need for video-rate confocal microscopy as it can be applied to their NIH-funded research programs. No such instrumentation is currently available at Harvard Medical School or its affiliated hospitals. The video-rate confocal microscopy system must include the following critical features: (1) An intravital microscopy facility for the use of live animals; (2) A laser confocal microscope system capable of rapid scanning and suitable for DIC and fluorescence microscopy under flow conditions; (3) A computer workstation with imaging software for image acquisition and reconstruction; (4) Multiple fluorescent channels for co- localization experiments. The BioRad RTS2000 real-time laser confocal system, fitted to a Nikon E600FN microscope, will provide a group of investigators with instrumentation to significantly advance their fields.