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落射荧光显微镜系统。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快速收集在显微镜载物台上培养的细胞的低光荧光图像。它将包括一个倒置落射荧光显微镜，电动，编码阶段，高性能CCD相机，和计算机与软件。将用一种或两种与激酶、GTP结合肌动蛋白相关蛋白和微管蛋白连接的突变绿色荧光蛋白（GFP）嵌合体转染细胞。活细胞也将用识别细胞外表位的荧光团缀合抗体标记。Metamorph软件将用于在图像采集期间控制电动载物台、摄像机、快门和滤光轮。该软件还将用于处理和分析图像，以及进行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。这些实验将监测鸡胚胎模型发育期间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摄像机、落射荧光立体镜、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。NIH资助的研究人员的核心将使用这种仪器。显微镜将存放在Lombardi癌症中心显微镜和成像共享资源中。首席研究员是一名经过培训的细胞生物学家，自1991年以来一直担任该设施的主任，自那时以来发表了18篇利用荧光显微镜的论文。她在光学显微镜和电子显微镜水平上接受了广泛的显微镜培训。在1999年，共享资源被46名调查员代表超过16个部门在医学院和本科校园使用。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的共聚焦显微镜。这台显微镜目前已满负荷运转。除了导演，一个全职经理/显微镜手是在手，以协助用户。共享资源由癌症中心支持和赠款支持相结合来维护。约625平方米英尺该资源占用了实验室空间，并将容纳拟议的设备。用户以用户收费的形式贡献资源。一个咨询委员会每年举行一次会议，监督费用、政策，并为未来的增长提出建议。

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