This application is to purchase a modern laser scanning confocal microscope to serve primarily the needs of the Cell Biology Department, Duke University Medical Center. The only departmental confocal at present is a 10 year old Bio-Rad MRC 600, which has limited capabilities in several areas. The microscope of our choice is the new Zeiss LSM 510 META. The META emission detector provides a unique and powerful detection system that far surpasses conventional dichroic filters. The META detector is based on a diffraction grating that spreads the emission into a full spectrum, and a detector that records the full spectrum from each scanned point. This provides a unique capability to separate fluors with overlapping emission spectra. The user provides a signature spectrum of each individual fluor, and the instrument then mathematically separates the spectrum into the separate components. Triple images with cfp, gfp and yfp should be completely separated. This ability to mathematically separate overlapping fluors is unique to the Zeiss. The ability to record the full spectrum of each image point will be extremely powerful for Fluorescence Resonance Energy Transfer. Another specialty of the Zeiss is its """"""""physiology"""""""" software package, which provides a very user-friendly facility for Fluorescence Recovery after Photobleaching. All of these capabilities are addressed in proposed projects. We also propose to purchase a module for Fluorescence Correlation Spectroscopy (FCS). This module illuminates a tiny volume and measures the fluctuations of fluorescently labeled molecules. This provides a means to assay for the diffusion coefficient, and thereby the polymer size, of labeled macromolecules. This can be applied to study molecules in solution, but more importantly, when used in conjunction with the scanning confocal mode, can perform these measurements in cells or tissues. Projects for this module are proposed by two faculty from Cell Biology and two from Biochemistry. This will be the only FCS facility at Duke University, and we expect additional projects from across the campus.
