A Confocal Laser Scanning Microscope for Cellular and Molecular Imaging

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This proposal requests funds for a confocal laser scanning microscope system based on an infmity corrected Zeiss Axiovert microscope with both differential interference and fluorescence capabilities for high resolution imaging. The confocal microscope will have a Krypton/Argon laser, three photomultiplier tubes (PMT) and a motorized transmitted light PMT. This microscope will be used to image a variety of cellular and molecular structures in both dynamic and still specimens at high resolution and in 3-dimensional detail. Different recording devices are requested. An optical disc and a CD-ROM unit will store images digitally and a hard copy color printer will allow quick retrieval of images. Dynamic structures will be recorded by a Panasonic Optical Memory Disc Recorder This proposal comes from 19 scientists from 9 different Departments and three Colleges at North Carolina State University and one Department at Wake Forest University. To our knowledge there is no other confocal laser scanning microscope for precise high resolution imaging available on either campus. Most of the seven project leaders are plant biologists investigating basic cellular, developmental and molecular processes using a combinatorial approach involving genetic, molecular, biochemical, and cell biological techniques and all have a very real need of confocal microscopy. The projects include investigations of the relationships between cytoskeleton and cell signaling (especially Calcium ions) as it influences plant development (Boss, Allen, Davies, Muday), of the targeting of storage proteins in maize (Boston) of viral pathogenesis and transport (Robertson and Lommel), of nematode pathogenesis and root specific gene expression (Conkling), and of ferredoxin localization as well as nuclear scaffold attachment regions in plant cells (Thompson). In addition, Peretti will study plasmid transfer in biofilms. Another group of investigators, who initially will be minor users, include the following: Thr ee members of Zoology will study various aspects of cell signaling in epithelial cells (Black), neurons and glia (Grossfeld) and from pituitary glands of fish (Borski). Mahaffey (Genetics) will study segmentation in Drosophila larvae and Weissinger will investigate fungal pathogenesis in transgenic plants. Foegeding (Food Science) will study biorheological properties of biopolymer gels and Laster (Microbiology) will investigate tumor induced apoptosis. Muday will study auxin transport in plants. Finally in Botany, Burkholder will study dinoflagellate life cycles. It is essential for many of these studies that fluorescent molecules or structures be precisely localized and resolved. The unique ability of the confocal microscope to make very thin optical sections at high resolution has opened new frontiers in cell biology in the last ten years, and this group of scientists has realized the opportunities available to greatly enhance their research efforts by applying the newer methods of molecular staining with confocal imaging and image analysis and quantification. North Carolina State University and the Department of Botany have made a very real commitment to strengthen cell biology and cellular imaging. This interest in imaging was driven in part by a need of the biotechnology and molecular biology oriented faculty to incorporate advanced imaging in their work. For example, it is now possible to use molecular biology techniques to express transgenically fluorescent probes such as Green Fluorescent Protein in plants. Several cell biologists have recently been hired in Botany and Zoology. A Cellular, Molecular and Developmental Biology Program is in development and will include faculty from many diverse Departments. Nina Allen will direct the Cellular and Molecular Imaging Center (CMIC) to be housed in the Botany Department in the area being renovated for Allen and Davies. Several high resolution video microscopes have been purchased with set-up money provided by NCSU to Allen. Wendy Boss has obtained funds to purchase a cooled-CCD imaging system that will be housed in CMIC. These microscopes in combination with the confocal microscope will allow great flexibility in imaging capabilities available in the Center and should greatly strengthen many areas of research at the University.