August 12, 1996: 12th International Biuophyics Congress, Amsterdam, The Netherlands. Non-linear laser microscopy based on multi-photon molecular excitation has reached a user-friendly maturity that offers a powerful tool for cellular and molecular biophysics. Intrinsically 3-d resolved microscopy for fluorescence imaging, photochemical micropharmacology and measurements of molecular dynamics are provided with minimal photodamage in living cells by simultaneous absorption of two or more red/infrared photons from the 100 MHz train of 100fs pulses of mode locked lasers. Two photon excitation spectra often show their largest cross sections at wavelengths far shorter than twice the one photon absorption peaks thus providing convenient red light excitation of many of the familiar visible absorbing as well as UV absorbing fluorophores. Three photon excitation provides convenient imaging of tryptophan, and polypeptide hormones and neurotransmitters that absorb below 300nm. Our three-photon and two photon excitation cross section data and physical theory of multi-photon excitation guides experimental design. Recent developments to be illustrated include sensitivity to detect single fluorophores in solution submicron 3-d resolved Ca activity ratio imaging at speeds up to 45 microseconds per pixel, neurotransmitter release by cage activation, and applications of multicolor fluorescent markers for simultaneous imaging of 4 colors with a single excitation wave length, quantitative imaging with biochemical indicators, 3-d resolved imaging of tissue autofluorescence and green fluorescent protein genetic markers, and deep tissue fluorescence imaging. Supported at DRBIO by NIH (RR04224 and RR07719) and NSF (BIR 9419978) Invited Lectures and Consultations: Prof. W. W. Webb 2 listings are occasionally listed per page, to save space.
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