FLUORESCENCE IMAGING OF LIVE CELLS AND TISSUES

点击翻译按钮获取中文摘要

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.