Funds are requested by the Emory University Biology Department for the purchase of a confocal microscope. The purchase will include a BioRad MRC-1000 confocal laser scanning imaging system and a Nikon Optiphot-2 microscope component. This system is unsurpassed in terms of specimen brightness, image resolution and ease of analysis. These capabilities are required by Biology Department labs that need to perform state of the art analyses of protein distribution in complex tissues. Principally four NSF-funded labs will use the equipment for research and training activities, although it is anticipated that wider departmental use will occur periodically. Three of the labs are investigating various aspects of insect neurogenesis (Ellis, Siegler and Yedvobnick) while the fourth lab is investigating sperm morphogenesis and migration in C. elegans (L'Hernault). The Ellis lab will utilize confocal microscopy to characterize the spatial distribution of the extramacrochaetae (emc) protein during embryonic development. Originally characterized for its role in achaete-scute regulation during sensory organ development, emc is now known to function more widely during development. Preliminary studies suggest that emc is expressed throughout the embryonic germ layers. The precise relationship between emc expression and achaete-scute expression will be characterized in the embryo. The Siegler lab will use confocal microscopy in conjunction with immunohistochemistry to identify neurons of known birthdate according to type, and thus test hypotheses about birth order within identified CNS lineages of the grasshopper. The Yedvobnick lab studies lateral inhibition and associated signal transduction to the nucleus during embryonic neurogenesis in Drosophila. They will utilize the equipment to investigate cytoplasmic-nuclear partitioning of the product of the neurogenic locus mastermind. They will also investigate potential physical interactions between cytoplasmic mastermind protein and the transmembranous Notch protein via colocalization assays. The L'Hernualt lab studies sperm morphogenesis that initiates as a multicellular syncitium. Confocal immunofluorescence will allow use of available antibodies to localize Golgi-derived membrane proteins within this complex three dimensional array. In addition, wild type and mutant sperm motility within the hermaphrodite reproductive tract will be examined by confocal microscopy, using already developed fluorescent tagging techniques. It is clear that conventional fluorescent microscopic techniques do not provide sufficient spatial detail to study these C. elegans sperm phenomena. The equipment will also be used extensively for the training of undergraduate and graduate students. The Biology Department has a sophisticated program for involving undergraduates in research. Beyond independent research Emory undergraduates are offered advanced project labs in Genetics, Neurobiology and Physiology; Development is planned for next year. In addition to academic year projects, summer research programs funded from NSF REU and the Howard Hughes Medical Institute bring in students from Emory and many other institutions. Although undergraduate use of the equipment will mostly be restricted to research purposes we anticipate incorporation of confocal microscopy demonstrations into the advanced project labs. Graduate students from programs including Neuroscience, Cell and Developmental Biology, and Genetics and Molecular Biology often rotate through Biology Department labs and several have joined the NSF-funded labs described above. Their utilization of the equipment should greatly enhance the quality of research output from the Biology Department.
