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. 1996 Oct 1;93(20):10763–10768. doi: 10.1073/pnas.93.20.10763

Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.

C Xu 1, W Zipfel 1, J B Shear 1, R M Williams 1, W W Webb 1
PMCID: PMC38229  PMID: 8855254

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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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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