Confocal Microscope

描述(由申请人提供)： 这一应用是为了使激光扫描共聚焦成像系统致力于在几个模型系统中对活细胞的动态进行时延或实时成像。该仪器将被整合到现有的生物成像设施中，该设施将搬进该部内新装修的空间。生物科学学院。成像系统将由主要用户的核心群体使用，并在有限的基础上(最多25%的时间)向新闻部内外的次要用户提供。三个主要用户(Dailey博士、Lilien博士和Soll博士)将使用该仪器来实现与NIH资助的项目相关的目标。戴利博士将使用延时成像技术，对携带绿色荧光蛋白(GFP)融合蛋白结构的大鼠脑片中的神经元进行成像，以研究突触形成和可塑性的机制。他将测试一种名为N-钙粘附素的黏附分子调节突触形成、可塑性和维持的假设。在另一个项目中，戴利博士将描述激活的小胶质细胞的运动行为，这是一种驻留在大脑中的细胞，在清除脑损伤后的死亡细胞和细胞碎片方面发挥着重要作用。同时利用双通道和三通道荧光时间推移成像来研究损伤脑组织中小胶质细胞与其他类型细胞之间的细胞-细胞相互作用。Lilien博士将在发育小鸡神经视网膜的过程中使用对转基因神经元的延时成像，以确定黏附分子如何影响轴突和树突生长的动态模式。这些研究将使用微扰剂测试不同蛋白质结构域的功能假说，这些微扰剂影响对N-钙粘蛋白功能至关重要的特定蛋白质-蛋白质相互作用，以及N-钙粘蛋白和b1-整合素之间的串扰。索尔博士将对细胞运动过程中细胞骨架蛋白的动态变化和活的网柄苔藓中的趋化作用进行分析。绿色荧光蛋白标记的蛋白质，包括肌动蛋白、肌球蛋白I和II，以及Wiscott-Aldrich蛋白SCAR，将在每隔2秒重建的细胞中使用该部门W.M.Keck图像分析设备开发的计算机辅助动态图像分析系统(2D和3D DIAS)进行分析。三位主要研究人员在时间推移共聚焦成像(Dailey)、黏附分子分子分析和cDNA工程(Lilien)以及细胞运动分析(Soll)方面的技术专长为项目之间的高度协同提供了确保其成功的保证。总之，这些研究将有助于理解细胞生长的分子机制，以及与人类正常组织发育和各种病理条件下的功能重塑相关的定向细胞运动。

DESCRIPTION (provided by applicant): This application is for a laser scanning confocal imaging system to be devoted to time-lapse or real-time imaging of the dynamic state of live cells in several model systems. The instrument will be integrated into an existing bio-imaging facility that will move into newly renovated space within the Dept. of Biological Sciences. The imaging system will be utilized by a core group of major users and will be available to minor users, both within and outside of the Department, on a limited basis (up to 25% time). The three major users (Drs. Dailey, Lilien, and Soll) will use the instrumentation to accomplish goals related to NIH-funded projects. Dr. Dailey will use time-lapse imaging of neurons in rat brain slices transfected with green fluorescent protein (GFP)-fusion protein constructs to study mechanisms of synapse formation and plasticity. He will test the hypothesis that an adhesion molecule, N-cadherin, regulates synapse formation, plasticity, and maintenance. In another project, Dr. Dailey will characterize the motility behaviors of activated microglia, a type of brain-resident cell that plays a major role in clearing dead cells and cellular debris following brain injury. Simultaneous 2- and 3-channel fluorescence time-lapse imaging will be utilized to study the cell-cell interactions between microglia and other cell types in injured brain tissues. Dr. Lilien will use time-lapse imaging of transfected neurons in developing chick neural retina to determine how adhesion molecules affect the dynamic patterns of axon and dendrite growth. These studies will test hypotheses on the functions of distinct protein domains using perturbational agents that affect specific protein-protein interactions important to N-cadherin function and to cross-talk between N-cadherin and b 1-integrins. Dr. Soll will perform analyses on dynamics of cytoskeletal proteins during cell motility and chemotaxis in live Dictyostelium. The dynamic remodeling of GFP-labeled proteins, including actin, myosins I and II, and the Wiscott-Aldrich protein SCAR, will be analyzed in cells reconstructed every 2 sec, using computer-assisted dynamic image analysis systems (2D and 3D DIAS) developed in the Department's W.M. Keck Image Analysis Facility. The technical expertise of the three primary investigators in time-lapse confocal imaging (Dailey), molecular analysis of adhesion molecules and engineering of cDNAs (Lilien), and motion analysis of cells (Soll) provides a high level of synergism between the projects that will assure their success. Together, these studies will contribute to an understanding of molecular mechanisms of cell growth and directed cell movements related to normal tissue development and to functional remodeling under a variety of pathological conditions in humans.