Quantitative Fluorescence Microscopy by Digital Image Analysis

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A video image memory system, acquistion procesor and digitizer, and a cursor generator will be acquired to complete a distributed, yet integrated, imaging facilty for the Center for Fluorescence Research in Biomedical Sciences. Interactive video image acquisition and analysis, and supermini computers for data acquisition and reduction are also part of the imaging facility. Four separate laboratories are networked to allow multiple user applications. A wide variety of projects in cell biology, cell physiology, developmental biology, regulatory biology, and biological chemistry will be supported by in the instrumentation. Among the projects to be supported are analyses of the growth factor stimulation of quiescent cells and macrophage chemotaxis. Ratio imaging will be used as a spectroscopic method for determining the spatial and temporal changes in (cellular) physiological parameters such as pH and pCa. Multiple parameter analysis of two or more separable fluorescent probes will also be used to correlate spatial and temporal dynamics of a variety of cellular funcitons. Three dimensional reconstruction will be carried out on actin networks in living cells. A method of two wavelength total internal reflection image analysis will be used to quantitate the distance from the cell substrate to selected molecules. Methods for mapping the spatial and temporal variations in fluorescence lifetime and fluorescence anisotropy of suitably labeled proteins in living cells will be developed. The first target for mapping with be calmodulin. Overall, the equipment, coupled with other resources in the Center, will permit state-of-the-art measurements greatly extending our understanding of cellular function.