The projects described by the users in this application share the common approach of using a combination of cell biology and quantitative optical techniques to explore a variety of processes. These investigators are most frequently dependent on the resolution of fluorescent indicators in living cells. The instrument chosen is a Leica CLSM confocal laser scan with a Fluorvert inverted microscope that will be 50% cost shared with the university. The design includes a complete two-channel detector system that allows simultaneous measurements of two different fluorescent labels in precise register for all image points. The high focusing accuracy provided will enable more precise measurements of cell volume than that afforded by other instruments and the Leica images are the best that we have observed. All of the users in this application have been funded by NIH for their work and for the experiments described in this proposal. The confocal microscope will bring a new dimension to their research by providing better resolution, removing uncertainty about co-localization, making measurements easier and in some cases, possible, for the first time. Several different types of measurements are described. In some cases the investigators will be exploring new territory in trying to make sense of a complicated pathogenesis pathway. In Forte's case, the ability to optically section entire Drosophila embryo is the best way to follow the developmental regulation of specific neural components. The Westbrook experiments will test a specific model for activation of a neural receptor by making simultaneous measurements of intracellular calcium levels and the activation state of the receptor -- difficult measurements to make without the resolving power of the confocal. Low's group currently makes many measurements using traditional fluorescence to compare co-localization of specific stains. The same measurements that take all day can be made more accurately using optical sectioning and the data can be stored on disk in just minutes. Thornberg's group need to make cell volume measurements that cannot be achieved in any other way than with a confocal.
