We are requesting equipment to complete a distributed, yet integrated, imaging facility for the Center for Fluorescence Research in Biomedical Sciences. Four separate laboratories will be networked to allow multiple user image acquisition and analysis based on primarily on our VICOM image processing system. Four major projects from the heart of the proposal, but other users from the Departments of Biological Sciences and Chemistry are expected. The growth factor stimulation of quiescent cells and macrophage chemotaxis will be the main biological studies. Project 1 focuses on ratio imaging as a spectroscopic methods to measure the spatial and temporal changes in physiological parameters such as pH an pCa. Project 2 is aimed at using multiple parameter analyses of 2 or more separable fluorescent probes to correlate the spatial and temporal dynamics of a variety of cellular functions. Project 3 will begin to perform 3-D reconstructions of actin networks in living cells and will quantify the distance from the cell substrate to selected molecules by 2 wavelength total internal reflection image analysis. Finally, Project 4 will begin to develop methods for mapping the spatial and temporal variations in fluorescence lifetime and fluorescence anisotrophy of suitably labeled proteins in living cells. The rotational diffusion of calmodulin will be the first target. Carnegie-Mellon has given the Fluorescence Center the resources to obtain all of the optical and electronic equipment for the time-resolved microscope.
