We are developing an optical workstation that will allow simultaneous multiphoton imaging and multiphoton uncaging. The workstation will have two ultra-fast, short-pulse, all-solid-state lasers to facilitate multiphoton excitation for 3-dimensionally localized uncaging and imaging of living cells and tissues. The uncaging laser will be a prototype Nd:YLF laser manufactured by Microlase, Ltd. which will produce 120 ferntosecond pulses at a 120 NfHZ rate at 650 nm with a mean power of >50 mwatts. The uncaging pattern (volume) will be user definable via software. The imaging laser is a tunable Spectra Physics Nfillenia/ Tsunami ferntosecond titanium sapphire laser with a mid mirror set which produces 60 ferntosecond pulses at a 80 MHz rate from 770 nm to 910 nm with a mean power >500 mwatts throughout the range. External pulse compensation will be implemented to maintain these ultra-short pulses at the sample. Laser beam scanning will be handled via NMC 1024 hardware and software. Each laser has its own independent scanning system. The scanned outputs will be combined and directed into a long working distance eyepiece beam expander attached to a Nikon Diaphot Quantum inverted microscope. Direct detection will be implemented via the Quantum Keller hole. Blue, green, and red emitting fluorophores in the range from 400 nm to 640 nm may be used with the system. The microscope will be contained within a three-gas incubator to facilitate live cell imaging viability as a function of excitation wavelength.
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