FLUORESCENCE IMAGING OF LIVE CELLS AND TISSUES

本提案要求拨款购买三套外延荧光显微镜系统。动态细胞成像提供了确定细胞结构和亚细胞蛋白定位之间的时间关系的可能性。因此，第一个系统将专门用于在显微镜台上培养的细胞的低光荧光图像的快速收集。它将包括一个倒置的外显荧光显微镜，电动的，编码的平台，一个高性能的CCD相机和带有软件的计算机。将细胞转染一个或两个突变的绿色荧光蛋白（GFP）嵌合体，这些嵌合体与激酶、GFP结合的肌动蛋白相关蛋白和微管蛋白相连。活细胞也将用识别细胞外表位的荧光基团偶联抗体进行标记。Metamorph软件将用于在图像采集期间控制电动舞台，相机，快门和滤光轮。该软件还将用于处理和分析图像，以及3D重建和反卷积。第二个显微镜系统将用于表达GFP或GFP嵌合体的组织的立体可视化。这些实验将在鸡胚模型发育过程中监测肿瘤细胞在组织中的侵袭/转移和蛋白表达。该系统的设备包括CCD相机、外显荧光立体镜、计算机和软件。第三种显微镜是许多用户共享的基本荧光显微镜的替代品。美国国立卫生研究院资助的研究人员的核心将使用该仪器。显微镜将被安置在伦巴第癌症中心显微镜和成像共享资源。首席研究员是一名训练有素的细胞生物学家，自1991年以来一直担任该设施的主任，从那时起发表了18篇利用荧光显微镜的论文。她在光学和电子显微镜水平上接受了广泛的显微镜训练。1999年，共有46名研究人员使用了共享资源，他们代表了医学院和本科校区16个以上的院系。自1999年5月以来，我们已经培训了40多名研究人员使用我们的新型共聚焦显微镜。这台显微镜目前已满负荷运行。除了主任之外，还有一名全职经理/显微镜师随时协助用户。共享资源是由癌症中心支持和赠款支持相结合来维护的。约625平方米。该资源占用了实验室空间的1英尺，并将容纳拟议的设备。用户以用户收费的形式对资源做出贡献。咨询委员会每年召开一次会议，监督收费、政策，并为未来的增长提出建议。

This proposal requests the funds to purchase three epi-fluorescence microscope systems. Dynamic cellular imaging affords the possibility of determining temporal relationships between cellular structures and subcellular protein localization. Thus, the first system will be specialized for fast collection of low light fluorescence images of cells cultured on the microscope stage. It will comprise an inverted epi-fluorescence microscope, motorized, encoded stage, a high performance CCD camera, and computer with software. The cells will be transfected with one or two mutant green fluorescence protein (GFP) chimeras linked to kinases, GTP-binding actin-associated proteins, and tubulin. Living cells will also be labeled with fluorophore-conjugated antibodies recognizing extracellular epitopes. Metamorph software will be used to control the motorized stage, camera, shutters, and filter wheels during image collection. This software will also be used to process and analyze the images, and for 3D reconstruction and deconvolution. The second microscope system will be used for stereoscopic visualization of tissues expressing GFP or chimeras of GFP. These experiments will monitor invasion/ metastasis of tumor cells in tissues and protein expression during development in a chicken embryo model. The equipment for this system comprises a CCD camera, epi-fluorescence stereoscope, computer and software. The third microscope is a replacement for a basic fluorescence microscope shared by many users. A core of NIH funded investigators will utilize this instrumentation. The microscope will be housed in the Lombardi Cancer Center Microscopy and Imaging Shared Resource. The Principle Investigator, a cell biologist by training, has been director of this facility since 1991, publishing 18 papers utilizing fluorescence microscopy since that time. She has extensive training in microscopy at both the light and electron microscopy levels. In 1999, the Shared Resource was utilized by 46 investigators representing more than 16 departments in the Medical School and Undergraduate campus. Since May 1999, we have trained over 40 investigators in the use of our new confocal microscope. This microscope is currently at full capacity. In addition to the director, a full time manager/microscopist is on hand to assist users. The Shared Resource is maintained by a combination of Cancer Center support and grant support. Approximately 625 sq. ft. of laboratory space is occupied by this resource and will house the proposed equipment. Users contribute to the resource in the form of user charge back fees. An advisory committee meets annually to oversee fees, policies, and to make recommendations for future growth.