FLUORESCENCE IMAGING OF LIVE CELLS AND TISSUES

活细胞和组织的荧光成像

• 批准号: 6291347
• 负责人: SUSETTE C MUELLER
• 金额: $ 22.15万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-15 至 2002-04-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6291347
• 关键词: bioassay; bioimaging /biomedical imaging; biomedical equipment purchase; biotechnology; chick embryo; computer program /software; fluorescence microscopy; image processing; neoplastic cell; tissue /cell culture

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.

该提案要求资金购买三个Epi-Fluorecence显微镜系统。动态细胞成像提供了确定细胞结构与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快速收集显微镜阶段培养的细胞的低光荧光图像。它将包括一个倒置的荧光显微镜，电动，编码阶段，高性能CCD摄像头以及带有软件的计算机。这些细胞将用与激酶，GTP结合肌动蛋白相关蛋白和微管蛋白相关的一个或两个突变绿色荧光蛋白（GFP）嵌合体转染。活细胞还将用识别细胞外表位的荧光团偶联抗体标记。 Metamorph软件将用于控制图像收集过程中电动舞台，相机，百叶窗和过滤轮。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。这些实验将监测组织模型中肿瘤细胞在组织中的侵袭/转移和蛋白质表达。该系统的设备包括CCD摄像头，Epi-Fluorecens Steaposcope，计算机和软件。第三个显微镜是替代许多用户共享的基本荧光显微镜。 NIH资助的研究人员的核心将利用这种仪器。显微镜将安置在伦巴第癌中心的显微镜和成像共享资源中。自1991年以来，主要研究者是一名细胞生物学家，一直担任该设施的主任，此后发表了18篇论文，从那时起就使用了荧光显微镜。在光和电子显微镜水平上，她都在显微镜下进行了广泛的训练。 1999年，共享资源被46位代表医学院和本科校园的16多个部门的调查人员利用。自1999年5月以来，我们已经培训了40多名研究人员，以使用新的共聚焦显微镜。该显微镜目前已满负荷。除主任外，全职经理/显微镜医生还在现场为用户提供帮助。共享资源是通过癌症中心支持和赠款支持的结合来维持的。大约625平方英尺的实验室空间被此资源占据，并将容纳拟议的设备。用户以用户充电费用的形式为资源做出了贡献。咨询委员会每年开会，以监督费用，政策并为未来增长提出建议。

项目成果

Tumor suppressor function of Syk in human MCF10A in vitro and normal mouse mammary epithelium in vivo.

• DOI: 10.1371/journal.pone.0007445
• 发表时间: 2009-10-15
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Sung YM;Xu X;Sun J;Mueller D;Sentissi K;Johnson P;Urbach E;Seillier-Moiseiwitsch F;Johnson MD;Mueller SC
• 通讯作者: Mueller SC