Multiphoton Molecular Excitation to Illuminate Non-linear Laser Microscopy

  • W. W. Webb
  • C. Xu
Conference paper
Part of the Springer Series in Chemical Physics book series (CHEMICAL, volume 62)


Molecular excitation by two or more infra red photons simultaneously absorbed from strongly focused 100 femtosecond mode locked laser pulses provides intrinsic 3-d resolution for fluorescence microscopy and photochemical micro pharmacology in living biological cells.1 Confinement of non-linear excitation to the focal volume illuminated by ~10 photons/nm s eliminates out of focus photobleaching and photodamage and provides intrinsic 3-d resolution. To facilitate this technology new methods have developed for accurate measurements of multi-photon excitation cross sections and spectra in the absence of adequate data to guide non-linear laser microscopy applications.2,3 Absolute cross section measurements have been facilitated by development of a method that takes advantage of the interference of excitation pulse trains that are time shifted by a conventional Michaelson interferometer to allow variable relative delay of two spatially superimposed beams. Two photon excitation with single mode CW lasers provides an independent absolute calibration of cross sections. Measurements of more than twenty useful two-photon excitation spectra (690–1050nm) have revealed some with large blue shifts of the excitation peak. Two photon excitation spectra of some asymmetric molecules superimpose on the one photon absorption spectra (with wavelength doubled) presumably due to relaxation of parity selection rules in these cases. Most of the tested fluorescent molecules show blue shifted excitation peaks with strongly enhanced cross sections exceeding those observed at twice the one photon absorption. No red shifts of the excitation spectra have been detected.


Excitation Spectrum Excitation Cross Section Focal Volume Photon Excitation Large Blue Shift 
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    W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).CrossRefADSGoogle Scholar
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    C. Xu and W. W. Webb, “Measurement of two-photon excitation cross-sections of molecular fluorophores with data from 690 nm to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).CrossRefADSGoogle Scholar
  3. (3).
    C. Xu, J. Guild, W. W. Webb, and W. Denk, “Determination of absolute two-photon excitation cross-sections by in situ second-order autocorrelation,” Opt. Lett. 20, 2372–2374 (1995).CrossRefADSGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1996

Authors and Affiliations

  • W. W. Webb
    • 1
  • C. Xu
    • 1
  1. 1.Applied PhysicsCornell UniversityIthacaUSA

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