Upgrade Existing Multiwell, Fluorescence Timelapse Imaging Workstation to Confoca

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DESCRIPTION (provided by applicant): Our original epifluorescence timelapse system was purchased in 2001 (Shared Instrumentation grant 1 S10 RR15768-01, "Fluorescence Imaging of Live Cells and Tissues", principal investigator: Mueller). It is critical need of upgrading to fulfill requirements for multicolor, multiwell, timelapse imaging for a core of nine NIH funded investigators. Although the Metamorph software and computer have been upgraded once using Lombardi Comprehensive Cancer Center Funds (LCCC), and a screening software module was purchased jointly by two Georgetown investigators in 2006 (Mueller and Elmendorf), this instrument is not confocal. The multiwell, automated imaging feature makes possible multiple, parallel experiments over hours or days and dramatically increases research efficiency and the power to compare treatment modalities under identical culture conditions. Past applications include cell migration assays, long term imaging to document changes in tumor cell behavior or in subcellular localization of GFP-chimeric proteins, and drug screening. The system consists of a Nikon Eclipse TE300 inverted epi-fluorescence microscope, ORCA-ER monochrome cooled CCD 12-bit camera, and MetaMorph image acquisition and analysis software version 7 with the Multiwell Screening and Analysis software module, a Sutter Instruments DG-4 high speed wavelength switcher for multi-color, live imaging, Prior ProScan motorized x,y,z stage controller, bright field and fluorescence shutters and filter wheels, and an environmental chamber. We request funds to convert this system to a confocal system with diode lasers, an EM-CCD camera for increased sensitivity, and a fast emission filter wheel. From 2004-2007, 18 investigators have utilized this instrument over 2230 hours. Four papers have been published, two submitted, and several are in preparation utilizing this microscope system. It is housed in the Microscopy and Imaging Shared Resource of the Lombardi Comprehensive Cancer Center (LCCC) which is directed by Dr. Mueller, a Professor of Oncology. The manager for this facility is Dr. Brian Koss. Drs. Xu and Govindan participate by directing the TIRF/ multiphoton component and education/ training, respectively. This facility is funded by LCCC, chargeback fees, and the LCCC Cancer Center Support Grant (2P30-CA-51008). PUBLIC HEALTH RELEVANCE: This proposal for funds to purchase a laser confocal upgrade to a research microscope will primarily support research at the Vincent T. Lombardi Cancer Center, a designated NIH Comprehensive Cancer Center and other investigators in the Georgetown University Medical Center and Main Campus. This laser confocal upgrade will facilitate the identification of cellular locations of proteins and subcellular structures that may cause tumor cell invasion and angiogenesis, development of hypertension, fungal and malarial infections and development of immunity, and effective drug targeting. This microscope will be placed in a pre-existing shared resource, used by a core of Lombardi investigators, and made available to other Georgetown investigators also supported by NIH-funded grants.