The specific aims of the Image Analysis Facility core are to provide: (1) specimen preparation and imaging of fixed material; (2) technical support for preparation and imaging of immunocyto-chemistry; (3) quantitative single and multiparameter steady-state analysis of vital fluorescence endpoints within living and/or stabilized cells and tissues; and, (4) quantitative single and multiparameter kinetic analysis of endpoints of cellular hemostasis mechanisms. The Image Analysis core is centrally located in the Veterinary Medical Administration building. The primary functions of the core are to provide access to microscopy and image analysis services for the evaluation of cellular hemostasis. Particular emphasis is placed on aims 3 and 4. These two aims are achieved using one of five fluorescence instruments: Meridian ACAS Ultima Interactive Laser Cytometer/Scanning Laser Confocal Microscope, Meridian InSIGHT Point Laser Scanning/Confocal Microscope, Scanalytics CELLscan Fluorescence Deconvolution Workstation, Photon Technologies International Fluorescence Ration Spectrometer, and Zeiss PMIII Light Microscope. Using these instruments, the Image Analysis core personnel have developed and/or adapted commercially available fluorescence probes and naturally occurring fluorescent molecules to monitor the following endpoints in cultured cells: (1) generation of reactive oxygen species; (2) analysis of intracellular pH; (3) determination of mitochondrial membrane potential; (4) detection of intracellular Ca2+ content, Ca2+ fluxes, intrinsic and induced Ca2+ oscillations; analysis of other ions such as Mg2+, Na+, K+, Zn2+, and Pb2+ Ca2+ interactions; (5) detection and quantitative assessment of gap junction mediated intracellular communication and metabolic cooperation; (6) analysis of plasma membrane integrity and membrane potential; (7) analysis of lipid and protein mobility in membranes (FRAP); (8) analysis of DNA content, unscheduled DNA synthesis, and quantitation of apoptosis; quantitative analysis of glutathione in single cells or in a cell population; (9) analysis of glutathione-S-transferase activity; (10) analysis of mixed function oxidase activity within specific fluorescence substrates; (11) analysis of (MDR-1 gene expression) p-glycoprotein induction/ expression; (12) analysis of cellular uptake, partitioning, extrusion, and/or metabolism of selected toxicants which are inherently fluorescent; (13) analysis of protein expression and cytoplasmic trafficking of selected proteins; (14) vital imaging of organelles for co-localization studies; (15) activation of """"""""caged"""""""" probes (e.g., caged second messengers, neurotransmitters); (16) analysis of endocytotic activity; and, (17) analysis of green fluorescent protein (GFP) as a reporter of gene expression and protein localization. The majority of these applications have been developed in collaboration with Center investigators and new applications are under development. Forty seven references which document the development or application of these methods by core personnel or Center investigators. The core also has micromanipulation and microinjection equipment and methods for cloning individual cells selected in vital assays by laser cytometry. Efforts are in progress to expand services such as the use of a square wave pulse generator to facilitate introduction of xenoproteins into cells or electroinsertion into plasma membranes for functional analyses using non-invasive imaging technologies. The application lists 14 users of this core among the Center?s investigators and at least 17 grants of those investigators that will be supported by this core. This facility core provides its services at a nominal cost to users and has acquired one new instrument every other year over the last nine years. The income from the user fees is used to cover the cost of supplies requests to use the core instruments and services must be made in writing. In general requests are handled on a first-come, first-serve basis.
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