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Applied Optics

Applied Optics

APPLICATIONS-CENTERED RESEARCH IN OPTICS

  • Vol. 37, Iss. 31 — Nov. 1, 1998
  • pp: 7352–7356

Two-Photon Fluorescence Excitation Cross Sections of Biomolecular Probes from 690 to 960 nm

Marius A. Albota, Chris Xu, and Watt W. Webb  »View Author Affiliations


Applied Optics, Vol. 37, Issue 31, pp. 7352-7356 (1998)
http://dx.doi.org/10.1364/AO.37.007352


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Abstract

We report on two-photon fluorescence excitation (TPE) action cross sections for five widely used molecular fluorophores. Measurements were performed by use of ultrashort (∼100-fs) Ti:sapphire pulsed excitation over the range 690–960 nm. TPE spectra were obtained by comparison with a fluorescein calibration standard. Large cross sections were found for the cyanine reagent Cy 3 (∼140 GM) and for Rhodamine 6G (∼150 GM), both at 700 nm [1 GM = 10<sup>−50</sup> (cm<sup>4</sup> s)/photon]. Several fluorophores show interesting and desirable blue shifts with respect to twice the one-photon absorption wavelength. Fluorophore fluorescence intensities showed no significant departure (∓4%) from quadratic illumination power dependence, indicating genuine two-photon processes. Implications of these measurements for two-photon laser-scanning microscopy are discussed.

© 1998 Optical Society of America

OCIS Codes
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(190.1900) Nonlinear optics : Diagnostic applications of nonlinear optics
(190.4180) Nonlinear optics : Multiphoton processes

Citation
Marius A. Albota, Chris Xu, and Watt W. Webb, "Two-Photon Fluorescence Excitation Cross Sections of Biomolecular Probes from 690 to 960 nm," Appl. Opt. 37, 7352-7356 (1998)
http://www.opticsinfobase.org/ao/abstract.cfm?URI=ao-37-31-7352


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References

  1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
  2. M. Göppert-Mayer, “Über Elementarakte mit zwei Quantensprüngen,” Ann. Phys. 9, 273–295 (1931).
  3. C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).
  4. C. Xu, W. Zipfel, J. Shear, R. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. 93, 10,763–10, 768 (1996).
  5. 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. 21, 2372–2374 (1995).
  6. J. B. Guild, C. Xu, and W. W. Webb, “Measurement of group delay dispersion of high numerical aperture objective lenses using two-photon excited fluorescence,” Appl. Opt. 36, 397–401 (1997).
  7. J. N. Demas and G. A. Crosby, “The measurement of photoluminescence quantum yields,” J. Phys. Chem. 75, 991–1024 (1971).

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