Abstract

We determined the group-delay dispersion (GDD) of five microscope objectives by measuring the second-order autocorrelation at the focal points of the objectives with two-photon excited fluorescence as the power square sensor. We found that typical microscope lens systems introduce significant GDD (2000–6500 fs2). The third-order dispersion determined for these objectives limits the minimum obtainable pulse width at the focal point of an objective to 20–30 fs if not compensated. No significant chromatic aberration or higher-order dispersion effects were found for any of the optical components measured within the wavelength range of 700–780 nm and for pulse widths greater than 50–60 fs.

© 1997 Optical Society of America

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References

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    [CrossRef] [PubMed]
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    [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
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1995 (3)

1994 (2)

R. M. Williams, D. W. Piston, W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8, 804–813 (1994).
[PubMed]

D. W. Piston, M. S. Kirby, C. Heping, W. J. Lederer, W. W. Webb, “Two-photon-excitation fluorescence imaging of three-dimensional calcium-ion activity,” Appl. Opt. 33, 662–669 (1994).
[CrossRef] [PubMed]

1993 (1)

1990 (1)

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

1987 (1)

1985 (1)

1984 (1)

1980 (1)

1967 (1)

J. A. Giordmaine, S. L. Rentzepis, S. L. Shapiro, K. W. Wecht, “Two-photon excitation of fluorescence by picosecond light pulses,” Appl. Phys. Lett. 11, 216–218 (1967).
[CrossRef]

Becker, P. C.

Brakenhoff, G. J.

Brito Cruz, C. H.

Denk, W.

C. Xu, J. B. Guild, W. W. Webb, W. Denk, “Determination of absolute two-photon excitation cross-section by in situ second-order autocorrelation,” Opt. Lett. 20, 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]

W. Denk, D. W. Piston, W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1995), pp. 445–458.
[CrossRef]

Diels, J.-C. M.

Fontaine, J.

Fork, R. L.

Giordmaine, J. A.

J. A. Giordmaine, S. L. Rentzepis, S. L. Shapiro, K. W. Wecht, “Two-photon excitation of fluorescence by picosecond light pulses,” Appl. Phys. Lett. 11, 216–218 (1967).
[CrossRef]

Gordon, J. P.

Guild, J. B.

Heping, C.

Kempe, M.

Kirby, M. S.

Lederer, W. J.

Loudon, R.

R. Loudon, The Quantum Theory of Light (Clarendon, Oxford, 1983), p. 82.

Marcuse, D.

Martinez, O. E.

McMichael, I. C.

Min, G.

G. Min, T. Tannous, C. J. R. Sheppard, “Three-dimensional confocal fluorescence imaging under ultrashort pulse illumination,” Opt. Commun. 117, 406–412 (1995).
[CrossRef]

Muller, M.

Piston, D. W.

D. W. Piston, M. S. Kirby, C. Heping, W. J. Lederer, W. W. Webb, “Two-photon-excitation fluorescence imaging of three-dimensional calcium-ion activity,” Appl. Opt. 33, 662–669 (1994).
[CrossRef] [PubMed]

R. M. Williams, D. W. Piston, W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8, 804–813 (1994).
[PubMed]

W. Denk, D. W. Piston, W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1995), pp. 445–458.
[CrossRef]

Rentzepis, S. L.

J. A. Giordmaine, S. L. Rentzepis, S. L. Shapiro, K. W. Wecht, “Two-photon excitation of fluorescence by picosecond light pulses,” Appl. Phys. Lett. 11, 216–218 (1967).
[CrossRef]

Rudolph, W.

Shank, C. V.

Shapiro, S. L.

J. A. Giordmaine, S. L. Rentzepis, S. L. Shapiro, K. W. Wecht, “Two-photon excitation of fluorescence by picosecond light pulses,” Appl. Phys. Lett. 11, 216–218 (1967).
[CrossRef]

Sheppard, C. J. R.

G. Min, T. Tannous, C. J. R. Sheppard, “Three-dimensional confocal fluorescence imaging under ultrashort pulse illumination,” Opt. Commun. 117, 406–412 (1995).
[CrossRef]

Simoni, F.

Squier, J.

Strickler, J. H.

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

Tannous, T.

G. Min, T. Tannous, C. J. R. Sheppard, “Three-dimensional confocal fluorescence imaging under ultrashort pulse illumination,” Opt. Commun. 117, 406–412 (1995).
[CrossRef]

Webb, W. W.

C. Xu, J. B. Guild, W. W. Webb, W. Denk, “Determination of absolute two-photon excitation cross-section by in situ second-order autocorrelation,” Opt. Lett. 20, 2372–2374 (1995).
[CrossRef]

R. M. Williams, D. W. Piston, W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8, 804–813 (1994).
[PubMed]

D. W. Piston, M. S. Kirby, C. Heping, W. J. Lederer, W. W. Webb, “Two-photon-excitation fluorescence imaging of three-dimensional calcium-ion activity,” Appl. Opt. 33, 662–669 (1994).
[CrossRef] [PubMed]

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

W. Denk, D. W. Piston, W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1995), pp. 445–458.
[CrossRef]

Wecht, K. W.

J. A. Giordmaine, S. L. Rentzepis, S. L. Shapiro, K. W. Wecht, “Two-photon excitation of fluorescence by picosecond light pulses,” Appl. Phys. Lett. 11, 216–218 (1967).
[CrossRef]

Williams, R. M.

R. M. Williams, D. W. Piston, W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8, 804–813 (1994).
[PubMed]

Xu, C.

Appl. Opt. (3)

Appl. Phys. Lett. (1)

J. A. Giordmaine, S. L. Rentzepis, S. L. Shapiro, K. W. Wecht, “Two-photon excitation of fluorescence by picosecond light pulses,” Appl. Phys. Lett. 11, 216–218 (1967).
[CrossRef]

FASEB J. (1)

R. M. Williams, D. W. Piston, W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8, 804–813 (1994).
[PubMed]

Opt. Commun. (1)

G. Min, T. Tannous, C. J. R. Sheppard, “Three-dimensional confocal fluorescence imaging under ultrashort pulse illumination,” Opt. Commun. 117, 406–412 (1995).
[CrossRef]

Opt. Lett. (5)

Science (1)

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

Other (2)

W. Denk, D. W. Piston, W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1995), pp. 445–458.
[CrossRef]

R. Loudon, The Quantum Theory of Light (Clarendon, Oxford, 1983), p. 82.

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Figures (3)

Fig. 1
Fig. 1

Experimental configuration for GDD measurements of microscope lenses. The experiments were performed with a mode-locked femtosecond Ti:sapphire laser. A prism pair pulse compression line, PC, was used to compensate for the GDD of the microscope objectives. A home-built Michelson interferometer provided the delay for the autocorrelation measurements. The laser pulse width was measured before the objective lens by a standard optical autocorrelator, and the laser bandwidth was monitored with a spectrometer. After two sequential beam expanders, BE (5× and 3×), the beam was ∼15 mm in 1/e2 diameter and completely overfilled the back aperture of each objective lens (5–10 mm diameter). The fluorescence from the 100-µm-thick 20-50-µM fluorescein sample was separated from the excitation by a long-pass dichroic mirror, DC, with reflectivity >95% for λ < 610 nm and detected by a photomultiplier tube, PMT. The PMT signal was recorded with a multichannel scalar.

Fig. 2
Fig. 2

Single-pass interferometric autocorrelation traces of mode-locked Ti:sapphire laser pulses (730 nm) measured with 2PE fluorescence of 20-µM fluorescein solution (aqueous NaOH, pH ∼ 13) and a Zeiss Plan-NeoFluar 1.4-NA oil-immersion objective. Trace a was measured with a near-transform-limited input pulse, ti, and trace b with optimum compensation for the dispersion of the optical system; c is an expanded version of b. The traces have been normalized with the baseline fluorescence to show the 8:1 peak-to-background ratio. FWHM pulse widths at the objective focal point, ts, were calculated from the measured autocorrelation FWHM, assuming a Gaussian pulse shape.

Fig. 3
Fig. 3

Relative 2PE fluorescence with and without pulse prechirp as a function of the input pulse-width at various levels of GDD. The curves are as follows: —, 500 fs2 GDD;––, 1000 fs2 GDD; –·–, 2000 fs2 GDD; –··–, 5000 fs2 GDD;···10,000 fs2 GDD; ·––· corresponds to the GDD of a 1.4-NA objective and eyepiece with 700-nm laser pulses (6500 fs2). The observed fluorescence loss for 60-fs excitation pulses is consistent with the predicted loss of 80%.

Tables (1)

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Table 1 Summary of GDD Data on Microscope Lenses as Measured by a Second-Order Interferometric Autocorrelation Functiona

Equations (2)

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F=12AδηCg20P2,
Fti, ϕ=12AδηCgpP2fti1+4 ln 2 ϕ/ti22.

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