We are requesting equipment to establish a multiuser confocal microscope facility. Each of the projects proposed in this application requires use of a confocal microscope, but none is currently available at the University of Colorado Medical School. Pilot studies with borrowed equipment have demonstrated directly the feasibility of most of the proposed projects. There are two essential components to the proposed facility: a laser scanning confocal microscope (LSCM) and a broadband confocal attachment for existing microscopes. Each type of microscope has certain advantages compared to the other; both types will be necessary to perform the proposed projects. The LSCM will be located in a dedicated central space which will be fully renovated through a $24,000 institutional commitment. The broadband confocal attachment and imaging workstation will be mobile, and so can be housed in a user's own laboratory. The devices will initially be shared by nine users from four different departments of the Medical School. Administrative provisions will encourage the addition of new users. Every user is currently funded by at least one PHS research grant; the total number of PHS research grants currently awarded to all participating investigators is 13. To maximize the availability of the confocal microscopes, we will transfer acquired images to other workstations (already available) for processing and analysis, if necessary. This arrangement will keep the confocal computers free for image acquisition, and avoid the bottleneck created by image processing and analysis. Most projects involve fluorescence microscopy of relatively thick preparations; both fixed (sectioned and whole mount) and living materials will be used. The key advantage of the confocal microscope is its ability to exclude photons emitted from above and below the plane of focus, thereby permitting resolution of structures which would otherwise be blurred by background illumination. This optical sectioning allows three-dimensional reconstructions from images obtained in multiple focal planes. In summary, the scientific projects, administrative structure, and institutional commitments will provide a flexible, time saving, and cost effective solution to the problem of meeting the research goals of multiple users.
