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 = 10-50 (cm4 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

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References

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  1. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
    [CrossRef] [PubMed]
  2. M. Göppert-Mayer, “Über Elementarakte mit zwei Quantensprüngen,” Ann. Phys. 9, 273–295 (1931).
    [CrossRef]
  3. C. Xu, 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).
    [CrossRef]
  4. C. Xu, W. Zipfel, J. Shear, R. Williams, W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. 93, 10,763–10,768 (1996).
    [CrossRef]
  5. C. Xu, J. Guild, W. W. Webb, W. Denk, “Determination of absolute two-photon excitation cross sections by in situ second-order autocorrelation,” Opt. Lett. 21, 2372–2374 (1995).
    [CrossRef]
  6. J. B. Guild, C. Xu, 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).
    [CrossRef] [PubMed]
  7. J. N. Demas, G. A. Crosby, “The measurement of photoluminescence quantum yields,” J. Phys. Chem. 75, 991–1024 (1971).
    [CrossRef]

1997

1996

C. Xu, 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).
[CrossRef]

C. Xu, W. Zipfel, J. Shear, R. Williams, W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. 93, 10,763–10,768 (1996).
[CrossRef]

1995

C. Xu, J. Guild, W. W. Webb, W. Denk, “Determination of absolute two-photon excitation cross sections by in situ second-order autocorrelation,” Opt. Lett. 21, 2372–2374 (1995).
[CrossRef]

1990

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

1971

J. N. Demas, G. A. Crosby, “The measurement of photoluminescence quantum yields,” J. Phys. Chem. 75, 991–1024 (1971).
[CrossRef]

1931

M. Göppert-Mayer, “Über Elementarakte mit zwei Quantensprüngen,” Ann. Phys. 9, 273–295 (1931).
[CrossRef]

Crosby, G. A.

J. N. Demas, G. A. Crosby, “The measurement of photoluminescence quantum yields,” J. Phys. Chem. 75, 991–1024 (1971).
[CrossRef]

Demas, J. N.

J. N. Demas, G. A. Crosby, “The measurement of photoluminescence quantum yields,” J. Phys. Chem. 75, 991–1024 (1971).
[CrossRef]

Denk, W.

C. Xu, J. Guild, W. W. Webb, W. Denk, “Determination of absolute two-photon excitation cross sections by in situ second-order autocorrelation,” Opt. Lett. 21, 2372–2374 (1995).
[CrossRef]

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Göppert-Mayer, M.

M. Göppert-Mayer, “Über Elementarakte mit zwei Quantensprüngen,” Ann. Phys. 9, 273–295 (1931).
[CrossRef]

Guild, J.

C. Xu, J. Guild, W. W. Webb, W. Denk, “Determination of absolute two-photon excitation cross sections by in situ second-order autocorrelation,” Opt. Lett. 21, 2372–2374 (1995).
[CrossRef]

Guild, J. B.

Shear, J.

C. Xu, W. Zipfel, J. Shear, R. Williams, W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. 93, 10,763–10,768 (1996).
[CrossRef]

Strickler, J. H.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Webb, W. W.

J. B. Guild, C. Xu, 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).
[CrossRef] [PubMed]

C. Xu, W. Zipfel, J. Shear, R. Williams, W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. 93, 10,763–10,768 (1996).
[CrossRef]

C. Xu, 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).
[CrossRef]

C. Xu, J. Guild, W. W. Webb, W. Denk, “Determination of absolute two-photon excitation cross sections by in situ second-order autocorrelation,” Opt. Lett. 21, 2372–2374 (1995).
[CrossRef]

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Williams, R.

C. Xu, W. Zipfel, J. Shear, R. Williams, W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. 93, 10,763–10,768 (1996).
[CrossRef]

Xu, C.

J. B. Guild, C. Xu, 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).
[CrossRef] [PubMed]

C. Xu, W. Zipfel, J. Shear, R. Williams, W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. 93, 10,763–10,768 (1996).
[CrossRef]

C. Xu, 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).
[CrossRef]

C. Xu, J. Guild, W. W. Webb, W. Denk, “Determination of absolute two-photon excitation cross sections by in situ second-order autocorrelation,” Opt. Lett. 21, 2372–2374 (1995).
[CrossRef]

Zipfel, W.

C. Xu, W. Zipfel, J. Shear, R. Williams, W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. 93, 10,763–10,768 (1996).
[CrossRef]

Ann. Phys.

M. Göppert-Mayer, “Über Elementarakte mit zwei Quantensprüngen,” Ann. Phys. 9, 273–295 (1931).
[CrossRef]

Appl. Opt.

J. Opt. Soc. Am. B

J. Phys. Chem.

J. N. Demas, G. A. Crosby, “The measurement of photoluminescence quantum yields,” J. Phys. Chem. 75, 991–1024 (1971).
[CrossRef]

Opt. Lett.

C. Xu, J. Guild, W. W. Webb, W. Denk, “Determination of absolute two-photon excitation cross sections by in situ second-order autocorrelation,” Opt. Lett. 21, 2372–2374 (1995).
[CrossRef]

Proc. Natl. Acad. Sci.

C. Xu, W. Zipfel, J. Shear, R. Williams, W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. 93, 10,763–10,768 (1996).
[CrossRef]

Science

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

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Figures (2)

Fig. 1
Fig. 1

Plots of the TPE action cross sections (filled circles) for (a) dextran–fluorescein (dextran MW of 10,000) at a concentration of 123 μM and dissolved in H2O at pH ∼ 11 (compare the curve with the TPE cross sections for fluorescein listed in Table 1), (b) Cy 3 on a logarithmic scale at a concentration of 65 μM dissolved in Na2CO3 buffer at pH ∼ 9.3, and (c) Rhodamine 6G on a logarithmic scale at a concentration of 110 μM dissolved in MeOH. The TPE cross section is defined as η2σ2 (see text). 1 GM = 10-50 (cm4 s)/photon.

Fig. 2
Fig. 2

TPE action spectra (filled circles) compared with the OPA spectra (dashed curve) of DiOC5 at a concentration of 81 μM dissolved in MeOH. Vertical-axis units: arbitrary for the OPA spectra and GM for the TPE spectra. Horizontal-axis units: wavelengths as shown for the TPE spectra; the OPA spectra wavelengths have been doubled as plotted. The TPE spectrum of DiOC6 is identical to the DiOC5 TPE spectrum within the limits of the experimental error (see text).

Tables (2)

Tables Icon

Table 1 TPA Cross Sections of Fluorescein at Selected Wavelengthsa

Tables Icon

Table 2 Slope in the Logarithmic Plot of Fluorescence Versus the Incident Intensity at Several Excitation Wavelengths for DiOC5a

Equations (6)

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σ TPE = η 2 σ 2 .
F t = 1 2   ϕ η 2 N t ,
F t 1 2   ϕ η 2 σ 2 C   8 p f τ 8 P t 2 π λ / n .
F t cal F t new = ϕ cal η 2 cal σ 2 cal C cal P cal t 2 n cal ϕ new η 2 new σ 2 new C new P new t 2 n new ,
σ 2 new λ η 2 new = ϕ cal η 2 cal σ 2 cal λ C cal ϕ new C new P cal t 2 P new t 2 F t new F t cal n cal n new .
σ 2 new λ η 2 new = ϕ FL 0.9 σ 2 FL λ C FL P FL t 2 F t new ϕ new C new P new t 2 F t FL ,

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