With the current application, five faculty in the Division of Neurobiology at the University of Arizona seek to establish a state-of-the-art biological imaging facility to enhance their studies of the development and functional organization of experimentally favorable insect nervous systems. The Division's current Imaging Facility, staffed by a full-time Assistant Staff Scientist, has a Bio-Rad MRC-600 laser scanning confocal microscope, which the five Major Users of this proposal use heavily for NIH-funded research. The MRC-600 is inefficient compared to newer confocal systems and is so popular that one now has to sign up for time 3 - 4 weeks in advance, making the confocal microscope the rate-limiting step in most of our experiments. Moreover, some confocal-microscopy projects have advanced to the point where additional resolution, the capability to visualize fluorescently labeled structures across a broad range of brightness, and the capability to image living preparations over long time periods under very low light levels would enhance our efforts. Therefore, the PI proposes to upgrade the MRC-600 confocal microscope to a MRC-1024, and to purchase a deconvolution microscopy system. The confocal upgrade will provide a larger pixel array for larger fields of view, more filter sets and three photomultiplier tubes for improved detection of multiple fluorophores, and a more efficient computer and software package. Deconvolution, while superficially similar to confocal microscopy in its ability to produce fluorescence images of significantly higher resolution than a standard fluorescence microscope, differs from the confocal microscope in two important ways: rather than rejecting out-of-focus light with a pinhole in the detector, it collects as much light as possible from the objective lenses and uses it to mathematically reconstruct the bright object; and rather than using a photomultiplier tube, which is noisy and whose output is not linearly related to input, it uses a CCD camera for sensitive, linear reporting. Thus, the deconvolution system will meet many of our emerging needs.
