A Confocal Imaging Facility for Upright and Inverted Microscopy

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A confocal microscope imaging facility will be established for six developmental biologists investigating cellular structures, events, and gene expression patterns in early Xenopus laevis embryos, in developing Manduca sexta nervous systems, and in mammalian endothelial cell-culture preparations. The confocal system will be configured with 3 sensitive photomultipliers and two lasers producing four visible-light lines, and with a compound upright microscope equipped with long-working distance water-immersion objectives to facilitate in vivo experiments involving microinjection or micromanipulation. The PI (Danilchik) has used a confocal system with an inverted microscope for several years to study the development of early Xenopus embryos, particularly on the vegetal (underside) surface of the fertilized egg, where localized molecules are relocalized via interactions with a complex and dynamic cytoskeleton. Recent work has turned to cytoskeleton-membrane interactions on the animal (upper) surface of the egg during cytokinesis. Current research plans are to follow particle motions, membrane fusion events, and cytoskeletal reorganization in the animal surface of living embryos. Because amphibian embryos cannot develop normally when turned upside down, an upright confocal system will be vital for these studies. Three other users (Copenhaver, Kent, Morton) study aspects of neural development and neuronal signalling in the tobacco hornworm Manduca sexta: Copenhaver studies the guidance of neuronal migration in Manduca, and has developed a live preparation in which neurons migrate in plain view along the dorsal surface of the gut. Kent uses Manduca as a model system to study developmental changes in motor terminals and dendrites in large whole-mount preparations and to examine synaptic specializations and growth cone morphology of identified motor neurons in vitro. Morton is examining the regulation of cyclic GMP (cGMP) production by neuropeptides in Manduca and C. elegans, and will require confocal microscopy with multiple laser lines to demonstrate colocalization of two or more antigens in wholemount preparations. Christian will use the confocal system to study the subcellular localization of components of the TGFBeta signal transduction pathway in developing Xenopus early embryos. Farrell studies the role of extracellular matrix components in the process of angiogenesis, and is interested in developing confocal methods to screen cell lines transfected with regulators of integrin receptor expression. Acquisition of a state-of-the-art confocal system with multiple laser lines and enhanced photon detection will greatly enhance and expand our individual research efforts, and will give us the ability to develop crucial live-cell imaging techniques. The instrumentation will be available for graduate students throughout the year and undergraduates in summer programs.