CONFOCAL MICROSCOPY FACILITY FOR LIVING SPECIMENS

目的是确保最先进的共聚焦显微镜 研究活体生物标本的设备，范围从体内 制备物、体外多细胞组织、单细胞和 亚细胞水平。 美国国立卫生研究院资助的15个主要赠款/项目将 从所描述的九个主要项目中受益， 仪器仪表 这九个项目的目的是评估： 模拟失重（E.M.）突触小泡 胞吐作用（M. Hay）;体内和体外微血管通透性（V.H. Huxley）; Ca和K通道分布（A.W. Jones）;内皮和 微血管平滑肌Ca（M.H.）Laughlin）;血管细胞Ca 慢性冠状动脉闭塞（J. L帕克）;内毒素血症中的钙火花 肌细胞（L.J.Rubin）;糖尿病中的平滑肌Ca（M. Sturek）; 血管生长因子受体（R.L. Terjung）。我们的需求是：1） 调整显微镜系统，以使用活的体内、体外组织， 单大肠杆菌制剂，2）将共聚焦研究与 电生理和显微注射技术，3）有足够的时间 解决动态事件。 现有设施是 不适合我们的需要，因为：1）它主要是化学适应 固定的准备，2）它通常每周服务5个主要用户，因此 造成我们国家卫生研究院所需的大量时间严重短缺， 资助活标本的显微镜研究，以及3）低时间 分辨率（2秒/图像）。 总体组织计划是购买 a Noran Odyssey（采集33 ms/图像），用于体内和体外 多细胞组织研究，这是通过适应 双发射宽场落射荧光能力将 包括多功能性。 一个诺兰奥兹将使主要单一的 钙动力学的细胞成像在4毫秒/图像，500倍快于在 现有设施。使用拟议设施的调查人员已 在活体标本的显微镜研究方面有丰富的经验。 两个数据 网络上的分析站将提供进一步的定量 分子的共定位和邻近，共聚焦图像的比较 去卷积方法和三维重建。 技术 大学将提供两年的维护支持 这些正在进行的研究项目。 预计这些主要 用户将占总使用量的85%以上， 要求的设备。 剩余时间将提供给其他 生物医学领域的研究小组 这种共焦 设备将使全面实施的新颖和多功能的 建议的调整，并将大大提高这些研究 项目 我们的主要优势是整合技术研究 活标本在单细胞和多细胞水平。

The objective is to secure state-of-the-art confocal microscopy equipment to study living biological specimens ranging from in vivo preparations, to in vitro multicellular tissues, to the single cell and subcellular levels. Fifteen major NIH funded grants/projects will benefit from the nine major projects described that require this instrumentation. The aims of the nine projects are to assess: vascular Ca in simulated weightlessness (E.M. Hasser); synaptic vesicle exocytosis (M. Hay); in vivo and in vitro microvessel permeability (V.H. Huxley); Ca and K channel distribution (A.W. Jones); endothelial and smooth muscle Ca in microvessels (M.H. Laughlin); vascular cell Ca in chronic coronary occlusion (J.L Parker); Ca sparks in endotoxemic myocytes (L.J. Rubin); smooth muscle Ca in diabetes (M. Sturek); vascular growth factor receptors (R.L. Terjung). Our needs are to: 1) adapt microscope systems to use living in vivo, in vitro tissue and single coli preparations, 2) combine confocal studies with electrophysiology and microinjection techniques, 3) have sufficient time resolution to resolve dynamic events. The existing facility is inadequate for our needs because: 1) it mainly accomodates chemically fixed preparations, 2) it serves typically 5 major users per week, thus creating a severe shortage of large time blocks required for our NIH funded microscopy-based research on living specimens, and 3) low time resolution (2 s/image). The overall organizational plan is to purchase a Noran Odyssey (acquisition 33 ms/image) for in vivo and in vitro multicellular tissue studies, which is enabled by adaptation of the stage, etc. Dual emission wide-field epifluorescence capabilities will be included for versatility. A Noran OZ will enable primarily single cell imaging of Ca dynamics at 4 ms/image, 500 times faster than in the existing facility. The investigators to use the proposed facility have extensive experience in microscopic study of living specimens. Two data analysis stations on a network will provide further quantitation of colocalization and proximity of molecules, comparison of confocal images to deconvolution methods, and 3-dimensional reconstruction. Technical support will be provided by the University for two years for maintenance of these ongoing research programs. It is anticipated that these major users will represent greater than 85 percent of the total usage of the requested equipment. The remaining time will be provided to other research groups in the biomedical area on campus. This confocal equipment will enable full implementation of the novel and versatile adaptations proposed and will substantially enhance these research projects. Our major strength is integration of techniques to study living specimens at single cell and multicellular levels.