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Research Article

Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy

C Xu, W Zipfel, J B Shear, R M Williams, and W W Webb

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    PNAS October 1, 1996 93 (20) 10763-10768; https://doi.org/10.1073/pnas.93.20.10763
    C Xu
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    W Zipfel
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    J B Shear
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    R M Williams
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    W W Webb
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    Abstract

    Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's palette of chemical indicators, fluorescent markers, and native biological fluorophores, including NADH, flavins, and green fluorescent proteins, that are applicable to living biological preparations. More than 25 two-photon excitation spectra of ultraviolet and visible absorbing molecules reveal useful cross sections, some conveniently blue-shifted, for near-infrared absorption. Measurements of three-photon fluorophore excitation spectra now define alternative windows at relatively benign wavelengths to excite deeper ultraviolet fluorophores. The inherent optical sectioning capability of nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background and photodamage. Here, the measured nonlinear excitation spectra and their photophysical characteristics that empower nonlinear laser microscopy for biological imaging are described.

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    Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy
    C Xu, W Zipfel, J B Shear, R M Williams, W W Webb
    Proceedings of the National Academy of Sciences Oct 1996, 93 (20) 10763-10768; DOI: 10.1073/pnas.93.20.10763

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    Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy
    C Xu, W Zipfel, J B Shear, R M Williams, W W Webb
    Proceedings of the National Academy of Sciences Oct 1996, 93 (20) 10763-10768; DOI: 10.1073/pnas.93.20.10763
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