Multi-photon microscopy is perhaps the most important advance in fluorescence microscopy since the introduction of the confocal microscope. Multi-photon microscopy allows visualization deep into structures and permits this visualization to be carded out over extended periods of time with minimal damage to the specimen. Among the advantages provided by multi-photon microscopy are: (a) no out of focus bleaching of fluorophores; (b) deeper optical imaging, because of reduced out of focus fluorophore excitation and decreased scattering of incident wavelengths; and (c) reduced phototoxicity for living specimens. This application requests funds to purchase a: 1.) BioRad Radiance 2100 multi-photon System; 2.) Coherent Mira 900-F tuneable Ti:S laser and Verdi 8 watt ND:YVO4 laser; and 3.) Nikon E-800 upright microscope and a Nikon TE-2000U inverted microscope. The Radiance 2100 multi-photon system has been selected for many reason, among them being a scanhead that can be moved with relative ease from one microscope stand to another, allowing the imaging system to be used with an upright stand for in vivo imaging or with an inverted stand for in vitro imaging. This is an important consideration in this application, because the multi-photon system will be installed in a campus-wide microscopy facility and serve a wide-range of users with different research interests and needs. The Radiance 2100 multi-photon imaging system will be housed in the W.M. Kcck Laboratory for Biological Imaging. The Laboratory was established in 1993 with a gift from the W. M. Keck Foundation, and is equipped with a BioRad MRC 1024 confocal microscope; a single-photon microscope with a mixed gas, krypton/argon laser, capable of three-line excitation. The Keck Laboratory is actively managed, supported by user fees, and is the only confocal microscopy service resource on the UW-Madison campus. The multi-photon imaging system will be operated and maintained by the Keck Laboratory, and access to the imaging system will be open to all qualified users, following well-established procedures that have functioned successfully since the inception of the Laboratory. Currently, the Laboratory provides more than 50 faculty and their students in different areas of biology the opportunity to use confocal microscopy in their research. Among the broad research areas represented by these faculty and students are: agronomy, animal science, biomedical engineering, cell, developmental, and molecular biology, genetics, neuroscience, nutritional sciences, oncology, pathology, reproductive, cardiac, and muscle physiology, etc. The research programs of many of these users will be advanced significantly if a multi-photon imaging system also is available to them in the Keck Laboratory. The acquisition of the BioRad 2100 multi-photon imaging System and associated equipment, as described above, will complement the Kcck Laboratory's BioRad MRC 1024 single-photon confocal microscope, and it will open new avenues of investigation for biological scientists from across the UW-Madison campus.
