The goal of this project is to develop a user friendly nonlinear optical microscope to be housed in the Center for Biomedical Imaging Technology (CBIT) at the University of Connecticut Health Center. This will be a cooperative project between Health Center faculty, the CBIT staff, and the University of Connecticut Photonics Research Center. The CBIT and Photonics Research Center staff will work together to couple new laser technologies to a scanning confocal microscope. The Health Center faculty who currently use laser scanning confocal fluorescence microscopy will be able to explore innovative biological applications with this emerging technology. An integral component of our approach is the synthesis of new nonlinear optical dyes by the CBIT staff for specific labeling of biological molecules. Although confocal microscopy has proven to be a powerful technique, nonlinear or two-photon excitation of fluorophores avoids many of the inherent disadvantages of traditional fluorescence confocal microscopy. In particular, out of plane photobleaching, photodamage and background fluorescence are greatly reduced in nonlinear excitation. In addition, this method has greater sensitivity and affords better depth discrimination and light penetration into thick samples. These properties make nonlinear microscopy an excellent method for the study of living cells. There are currently seventeen academic laboratories at the Health Center who utilize confocal microscopy and this improved methodology is expected to be used by many of these groups. The research of Carson, Cowan, Jaffe and Terasaki, Koppel, and Loew are highlighted in this proposal; the nonlinear microscope will improve the quality and capabilities of their current experiments on a variety of living cells. Loew and CBIT will design and synthesize new dyes to be used in two photon fluorescence and second harmonic generation to measure membrane potential. The photophysics of these dyes will be charac terized with the new laser systems. In addition, some of the local pharmaceutical companies are active confocal users and are expected to takes advantage of the new instrument. The CBIT Bio-Rad MRC600 confocal microscope is being dedicated to the development of this instrument. The project will begin by modifying this microscope for nonlinear absorption and detection. High peak power pulsed lasers are required for nonlinear absorption and both commercial and custom laser systems will be utilized in this project. A commercial Titanium Sapphire femtosecond laser system will provide wavelength coverage from 720-900 nm for absorption from 360-450 nm. Concurrently the Photonics Research Center will develop user-friendly fiber-optic based modelocked lasers which will operate at 1064 nm and 1500 nm. These lasers will be integrated into the nonlinear microscope as the technology become available. The first generation of fiber lasers will initially be actively modelocked since this technology is expected to be straightforward to implement. The next generation will be passively modelocked and is expected to be very robust. Initially these lasers will provide pulse widths of less than 100 picoseconds and the developmental process will include efforts to further reduce these widths. In the long term, these lasers are expected to be more cost effective than Titanium Sapphire systems and easier for the casual user to operate. To provide a wide range of wavelengths, and thus usable fluorescent labeling dyes, the Titanium Sapphire and fiber lasers can be used together for sum-frequency excitation allowing coverage of a broad range of fluorescent probes absorbing from 350 - 750 nm. It is hope that this combination of fiber optic lasers and nonlinear microscopy will become a standard experimental method, much like laser scanning confocal microscopy has emerged as such in the last decade. Indeed, the advantages of 2-photon and other non-linear microscopies are so s ignificant that the development of reasonable lasers might very well breach the only barrier to having this new technology completely replace confocal microscopy.
