The requested funds will be used to upgrade a Bio-Rad MRC-600 confocal microscope. The items to be purchased are: a Krypton/Argon laser with fiber optic mount, improved photomultiplier tubes, a 60X 1.2 NA water immersion lens, and a computer for image processing. The new laser will dramatically improve the color separation in double label immunofluorescence experiments and allow triple label experiments to be performed. The fiber optic mount will decrease vibrations in the scan head and improve resolution at high magnification. The new photomultiplier tubes will allow the imaging of fainter objects and the water immersion lens will improve the quality of images from thick specimens. By having a second computer for off line processing, one researcher can be collecting data while a second is processing data. The equipment will be used in both cell biological and developmental experiments. The cell biological experiments center on the trafficking of molecules through the cell. Studies will be performed to understand how the Drosophila Wingless secreted protein is taken up by cells and the potential role of transcytosis in the formation of a gradient of Wingless protein. These experiments should lead to a better understanding of how pattern is generated in developing organisms. In other work the role of the Rab 7 protein in the endocytic pathway will be examined. The goal of this work is to understand how molecules are targeted to various organelles. The third main area of cell biological study is the use of cationic lipids for delivering molecules into cells. This technology will be used to study a variety of cellular processes. All these studies require co-localization experiments in which it is critical to maximize color separation. In addition, the ability to follow the subcellular distribution of molecules requires the best possible resolution and highest sensitivity. The Developmental studies are focused on Drosophila. In addition to the Wingle ss experiments described above, research is being conducted on the transcriptional regulation of leg patterning and the role of cell lineage in the patterning of the central nervous system. Both of these studies require an ability to carry out triple label experiments in which three molecules can be followed simultaneously. The goal of the leg patterning experiments is to understand how a gradient of positional information is interpreted and leads to the specification of individual cell fates. The neuronal lineage experiments will examine the relative contribution of lineage and positional information to the final phenotype of individual neurons. The Krypton/Argon laser is essential for the triple label experiments and the water immersion lens will allow neurons and their axonal projections to be followed throughout the embryo.
