DEVELOPMENT OF TWO PHOTON IMAGING SYSTEM FOR STUDY OF LIVING SPECIMENS

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We are exploring the potential applications of multiphoton imaging for biological or clinical research. Multiphoton imaging offers two significant advantages over confocal imaging: reduced total photobleaching (and hence phototoxicity) and increased depth of penetration within a specimen. We have developed a multiphoton system that features a compact, all-solid-state laser. The system also has an environmental chamber to facilitate studies of live specimens. Two lasers are utilized to provide comparisons between confocal imaging (one-photon excitation) and two and thr-ee-photon excitation images. The first laser is a standard argon ion laser which delivers 5 mwatts average power at 488 mn into the scanner. The second laser is an infrared, short-pulse (120fsec), solid-state laser manufactured by Microlase, Ltd. which produces a mean power of 480 mwatts at 1047 nm. The laser scanning system is based on a BioRad MRC 600 confocal system and is coupled to a Nikon Diaphot Quantum inverted microscope. Two detection options are available. The emission signal may be directed back into the scanhead and detected by the internal PMTs. Descanning the emission provides the option of introducing the "pinhole" into the imaging pathway when deemed necessary to trim the resolution. Otherwise, the emission signal can be passed directly through the "Keller" hole to a PNff mounted below the microscope. By detecting the emission directly, we have gained approximately 17 times more signal detection than descanning.