We request funding for the purchase of an integrated digital deconvolution microscope workstation to examine the expression and localization of proteins, chromosomes, organelles, viruses and cytoskeletal elements in fixed and living cells. With the new digital deconvolution microscope, researchers at Princeton University will be able to optically section biological specimens with unprecedented resolution (better than 0.2 micron) and clarity. The deconvolution microscope can achieve this outstanding level of resolution by using computational methods to digitally remove """"""""out-of focus"""""""" light from other focal planes. The optical slices can them be combined to produce highly accurate three-dimensional models of sub-cellular structures. Coupled to fast and extremely sensitive digital cameras (able to detect single photons), the deconvolution microscope will allow researchers to follow rapid dynamic processes within living cells using fast time-lapse imaging. This proposal details four major, and one minor, projects where the digital deconvolution will have a major impact. These projects include: (1) elucidation of the long range chromosome interactions that occur during site-specific mitotic recombination such as occurs during mating type interconversion in yeast, (2) the visualization of Herpes virus invasion and spreading in neuronal cells, (3) analyzing the roles of microtubule orientation and dynamics on nuclear orientation and migration using yeast as a model system, (4) visualization of telomeres and the nuclear envelope to examine the role of nuclear peripheral localization on the transcriptional repression that occurs at telomeres, and (5) analysis of the secretory pathway in live yeast cells to test different models of Golgi biogenesis. In each case, because of the extremely small size of the cells and/or of their internal structures, these projects require the unique optical sectioning power and resolution of the digital deconvolution microscope. By these means, the digital deconvolution microscope will provide outstanding research and educational opportunities on one of the most powerful optical microscopes available for use in the life sciences.
