This application requests funds to purchase a state of the art Applied Precision Delta Vision wide-field deconvolution microscope system. Live cell imaging is becoming an ever important aspect of biological studies for Salk Institute investigators. The requested system is specifically designed to provide high resolution analysis of living cells and tissues without the negative aspects of the laser illumination used in confocal microscopy. The proposed research involves tumor suppressor genes, genetic stability, protein trafficking, intranuclear structures implicated in cancer, viral invasion, cell motility, cytokinesis, and neuronal cell killing mediated by glutamate, Alzheimer's amyloid protein, and polyglutamate tracts in Huntington's disease. The majority of the studies require time lapse imaging, three-dimensional reconstruction, analysis of multiple cells using two or more fluorophores to enable co-localization, strict temperature control, and low intensity light to avoid photodamage and activation of cellular stress responses. The Salk Institute currently lacks an instrument with all of these functions. The requested DeltaVision system has been designed to meet all of the experimental criteria of the major user group. Deconvolution microscopy employs a standard epifluorescent microscope with highly optimized wide field illumination. Thus, high intensity laser illumination is not used avoiding photodamage which can harm living cells or activate stress responses, or photobleaching which can compromise quantitative analysis. Further, living cells can move during a time-lapse experiment, causing the region of interest to leave the illuminated plane, but the wide field and 3-D imaging capacity of the deconvolution system minimizes this problem. Finally, constrained iterative deconvolution is quantitative, conserves the maximum amount of data, affords high sensitivity and resolution with minimum illumination. Since excitation and emission filter wheels are used to select the proper wavelengths from a standard Hg-vapor lamp, new wavelengths can be added by simply adding the appropriate filters. This is in contrast to the limited excitation capabilities of confocal microscopy where restrictive laser lines limit the fluorophores that can be visualized, and excitation in the UV requires an extremely expensive UV-laser (which is also cytotoxic). The flexibility, high dynamic range and sensitivity, multiple cell sampling, and rapid three-dimensional imaging properties of the requested instrument will expedite completion of the proposed work which has important implications for cancer, viral pathogenesis, and nervous system diseases.
