This grant proposal seeks funding for a laser scanning confocal microscope to perform localization of a variety of proteins and cells using double and triple labeling of antigens. The research interests of the user group are diverse ranging from investigating cellular diversity within the gastrointestinal tract to cardiac physiology, but all have common interests in measuring the spatial apposition between cells, changes in protein expression during cellular transdifferentiation, and the localization of proteins within specific cell types. The research record of the user group is excellent. The user group has considerable experienced and has published extensively using confocal microscopy. However, because of limitations, age and extensive use of our current microscope (Biorad MRC 600) many of the morphological questions that have arisen during the course of current investigations cannot be adequately addressed. This is a serious limitation for the ongoing projects of the user group. Questions that need to be addressed include an examination of the spatial relationships between cells in heterogeneous tissues, the co- localization of organelles and proteins and translocation of proteins within cells. These are questions that all require double and triple labeling of antigen sites on the surface or within cells. The ideal instrument to facilitate rigorous completion of specific aims of already funded projects to incorporate a Zeiss LSM 510 laser confocal microscope. The Zeiss provides up to 4 confocal channel configurations for reflected light plus one for transmitted light (DIC), multi-tracking for critical flurochrome combinations, individual adjustable pinholes for correct 3-dimensional measurements, 12 bit data acquisition and 2048x2048 pixel resolution. The Zeiss offers the greatest integrated system available for these studies. Research at the University of Nevada has grown dramatically over the last number of years. The proposed user group of the Zeiss LSM 510 is highly competitive, representing some of the most successful investigators at the University of Nevada. The institution recognizes the quality of the user group by providing yearly support for a service contract. The PI has had considerable experience with confocal microscopy and will chair the internal advisory committee and oversee the daily operation of the instrument. The instrument will be housed in an existing NIH funded imaging core laboratory.
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