High-resolution visualization of cellular compartments and components is crucial to a wide variety of research projects at the Penn State College of Medicine, including co-localization studies, studies of macromolecular interactions using FRET analysis, and transport/kinetic studies of movement within cells. This proposal requests funds to replace our aged and no longer adequate Zeiss 210 upright confocal microscope with a new Leica TCS SP2 inverted microscope confocal system. The upgrading of our confocal microscopy instrumentation will enable us to add a crucial service in our shared Core Facilities which will be used by numerous NIH-funded investigators, twelve of whom are still included as the major users group in this proposal. Although the current confocal microscope is still adequate for very simple studies, and has produced images used in numerous publications from our researchers, the lack of a third laser line, the pack of 3D reconstruction software, the inability to keep images in registration when changing dichroic filters or changing to transmission optics, the cumbersome interface, and the poor service record for the 210 make it impossible to use for most current applications and research needs. For these reasons, our researchers have either had to go elsewhere for their confocal needs (1 100 mile drive to our State College campus), or find alternative, less-than-optimal solutions to addressing their research aims. Since the current confocal is known to be very difficult to use and often not functioning, this has also prevented new researchers, with new projects that would benefit from the use of a good confocal system, from establishing investigative systems using confocal microscopy. In addition to our own testing and extensive consultation with imaging scientists with imaging scientists concerning competing confocal systems (BioRad, Olympus, Nikkon, Zeiss and Leica), we have spoken at length with imaging experts at six different academic locations, and also investigated the possibility of upgrading our current system (Microcosm, Columbia, MD). The requested Leica TCS SP2 confocal system is a state-of-the-art system that will meet all of our current and foreseeable needs for confocal microscopy, and its tuneable emission detection allows both optimal separation of current fluors and the flexibility to adapt new fluorescent problems as they are developed. The requested instrument will provide crucial support to our major users group's projects, comprising 21 NIH grants totally over $2 million in current yearly direct costs, as well as aiding the development of new research projects in our larger research community.
