This proposal requests partial funding for a Microscopic Imaging Laboratory to be used by members of the Departments Molecular Cancer Biology of the Duke University Medical Center and Department of Botany of Duke University. The users already have a Zeiss AxioSkop microscope outfitted for fluorescence microscopy, which was purchased with funds from Duke University. This application proposes to purchase a Hamamatsu cooled charged coupled device (CCD) camera with a thermal printer and monitor that would provide the capability of high resolution and high sensitivity imaging. Accessories that would greatly enhance the flourescent capabilities of the instrument and the camera are also proposed. Moreover, a Z-axis stepper is proposed in order to be able to obtain 3D reconstructions. Two areas of great interest are represented by the work proposed here: first, the understanding of key regulatory events in the cell cycle of eukaryotes and second, the unraveling of the architecture of the eukaryotic nucleus. The work on the cell cycle is carried on by Drs. Lew, Garrett, Horowitz, Kombluth and Swenson. The relationship between regulatory events in the cell cycle and cellular morphology is studied by Dr. Lew using the baker's yeast Saccharomyces cerevisiae. Dr. Swenson is elucidating signalling pathways controlling entry into meiosis in surf clam and amphibian oocytes. Dr. Horowitz's laboratory focuses on the regulation of the cell cycle in mammalian cells. The regulation of DNA synthesis as an integral decision of cycling cells is investigated in Xenopous laevis eggs by Dr. Kornbluth. Work on nuclear architecture is ongoing in the laboratories of Drs. Garcia Blanco, Kohorn and Kornbluth. The project in Dr. Garcia Blanco's laboratory focuses on the relationship between nuclear architecture and the machineries that processes pre-rRNA and pre-mRNAs. Dr. Kohorn, in collaboration with Dr. Garcia Blanco, is investigating the nuclear architecture in Chlamydomonas. Th is organisms provides the possibility of doing biochemical and genetic analysis of nuclear functions and is much better than Saccharomyces cerevisiae for studies that visualize nuclear structure. These projects require precise image acquisition and storage systems. The continuation of the work requires a high sensitivity and high resolution camera to elucidate new cellular structures.
