Abstract

We report on a novel method designed for measuring two-photon action cross sections spectra in a single shot without tuning the excitation wavelength. Our technique is based on (i) using a nonlinear photonic crystal fiber to broaden the spectrum of the femtosecond excitation pulses and (ii) exploiting angular dispersion to focus different wavelengths to different lateral positions. As a result, two-photon fluorescence signal at different excitation wavelengths can be obtained simultaneously. As a proof of principle, the relative two-photon action cross sections of rhodamine green and DiI-C18 are measured over 740–860 nm range using fluorescein as a reference. Our results are in good agreement with that obtained using conventional tunable mode-locked laser.

©2006 Optical Society of America

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References

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    [Crossref]
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    [Crossref]
  4. R. Kappoor, C. S. Friend, and A. Patra, “Two-photon-excited absolute emission cross-sectional measurements calibrated with a luminance meter,” J. Opt. Soc. Am. B 20, 1550–1554 (2003).
    [Crossref]
  5. P. Kaatz and D. S. Shelton, “Two-photon fluorescence cross-section measurements calibrated with hyper-Rayleigh scattering,” J. Opt. Soc. Am. B 16, 998–1006 (1999).
    [Crossref]
  6. M. Schneideret al., “Two-Photon activation and excitation properties of PA-GFP in the 720–920-nm region,” Biophys. J. 89, 1346–1352 (2005).
    [Crossref] [PubMed]
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    [Crossref]
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  11. K. Shi, P. Li, S. Yin, and Z. Liu, “Chromatic confocal microscopy using supercontinuum light,” Opt. Express 12, 2096–2101 (2004).
    [Crossref] [PubMed]
  12. K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223, 83–87 (2006).
    [Crossref] [PubMed]
  13. G. P. Agrawal, Nonlinear Fiber Optics, 2nd ed. (Academic Press, London, 1995).
  14. C. Tunget al., “Effects of index-mismatch-induced spherical aberration on two-photon imaging in skin and tissue-like constructs,” in Multiphoton Microscopy in the Biomedical Sciences III, A. Periasamy and P. T. C. So, eds., Proc. SPIE 4963, 95–104 (2003).
    [Crossref]
  15. E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. 1. Conceptual basis and theory,” Biopolymers 13, 1–27 (1974).
    [Crossref]
  16. J. K. Ranka, R. S. Windeler, and A. J. Stentz, “Visible continuum generation in air-silica microstructure optical fibers with anomalous dispersion at 800 nm,” Opt. Lett. 25, 25–27 (2000).
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  17. D. Meshulach and Y. Silberberg, “Coherent quantum control of two-photon transitions by a femtosecond laser pulse,” Nature 396, 239–242 (1998).
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  18. J. P. Ogilvieet al., “Use of coherent control for selective two-photon fluorescence microscopy in live organisms,” Opt. Express 14, 759–766 (2006).
    [Crossref] [PubMed]

2006 (2)

K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223, 83–87 (2006).
[Crossref] [PubMed]

J. P. Ogilvieet al., “Use of coherent control for selective two-photon fluorescence microscopy in live organisms,” Opt. Express 14, 759–766 (2006).
[Crossref] [PubMed]

2005 (1)

M. Schneideret al., “Two-Photon activation and excitation properties of PA-GFP in the 720–920-nm region,” Biophys. J. 89, 1346–1352 (2005).
[Crossref] [PubMed]

2004 (1)

2003 (2)

C. Tunget al., “Effects of index-mismatch-induced spherical aberration on two-photon imaging in skin and tissue-like constructs,” in Multiphoton Microscopy in the Biomedical Sciences III, A. Periasamy and P. T. C. So, eds., Proc. SPIE 4963, 95–104 (2003).
[Crossref]

R. Kappoor, C. S. Friend, and A. Patra, “Two-photon-excited absolute emission cross-sectional measurements calibrated with a luminance meter,” J. Opt. Soc. Am. B 20, 1550–1554 (2003).
[Crossref]

2001 (1)

G. A. Blabet al., “Two-photon excitation action cross-sections of the autofluorescent proteins,” Chem. Phys. Lett. 350, 71–77 (2001).
[Crossref]

2000 (2)

1999 (1)

1998 (3)

1996 (2)

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “2-Photon Laser Scanning Fluorescence Microscopy,” Science 248, 73–76 (1990).
[Crossref] [PubMed]

1974 (1)

E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. 1. Conceptual basis and theory,” Biopolymers 13, 1–27 (1974).
[Crossref]

Agrawal, G. P.

G. P. Agrawal, Nonlinear Fiber Optics, 2nd ed. (Academic Press, London, 1995).

Albota, M. A.

Atkin, D. M.

Birks, T. A.

Blab, G. A.

G. A. Blabet al., “Two-photon excitation action cross-sections of the autofluorescent proteins,” Chem. Phys. Lett. 350, 71–77 (2001).
[Crossref]

Bouma, B. E.

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, “2-Photon Laser Scanning Fluorescence Microscopy,” Science 248, 73–76 (1990).
[Crossref] [PubMed]

Elson, E. L.

E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. 1. Conceptual basis and theory,” Biopolymers 13, 1–27 (1974).
[Crossref]

Friend, C. S.

Kaatz, P.

Kappoor, R.

Knight, J. C.

Konig, K.

K. Konig, “Multiphoton microscopy in life sciences,” J. of Microsc. 200, 83–104 (2000).
[Crossref]

Li, P.

Liu, Z.

K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223, 83–87 (2006).
[Crossref] [PubMed]

K. Shi, P. Li, S. Yin, and Z. Liu, “Chromatic confocal microscopy using supercontinuum light,” Opt. Express 12, 2096–2101 (2004).
[Crossref] [PubMed]

Magde, D.

E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. 1. Conceptual basis and theory,” Biopolymers 13, 1–27 (1974).
[Crossref]

Meshulach, D.

D. Meshulach and Y. Silberberg, “Coherent quantum control of two-photon transitions by a femtosecond laser pulse,” Nature 396, 239–242 (1998).
[Crossref]

Ogilvie, J. P.

Patra, A.

Ranka, J. K.

Russell, P. S

Schneider, M.

M. Schneideret al., “Two-Photon activation and excitation properties of PA-GFP in the 720–920-nm region,” Biophys. J. 89, 1346–1352 (2005).
[Crossref] [PubMed]

Shelton, D. S.

Shi, K.

K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223, 83–87 (2006).
[Crossref] [PubMed]

K. Shi, P. Li, S. Yin, and Z. Liu, “Chromatic confocal microscopy using supercontinuum light,” Opt. Express 12, 2096–2101 (2004).
[Crossref] [PubMed]

Silberberg, Y.

D. Meshulach and Y. Silberberg, “Coherent quantum control of two-photon transitions by a femtosecond laser pulse,” Nature 396, 239–242 (1998).
[Crossref]

Stentz, A. J.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “2-Photon Laser Scanning Fluorescence Microscopy,” Science 248, 73–76 (1990).
[Crossref] [PubMed]

Tearney, G. J.

Tung, C.

C. Tunget al., “Effects of index-mismatch-induced spherical aberration on two-photon imaging in skin and tissue-like constructs,” in Multiphoton Microscopy in the Biomedical Sciences III, A. Periasamy and P. T. C. So, eds., Proc. SPIE 4963, 95–104 (2003).
[Crossref]

Webb, R. H.

Webb, W. W.

Webb, W.W.

Windeler, R. S.

Xu, C.

Yin, S.

K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223, 83–87 (2006).
[Crossref] [PubMed]

K. Shi, P. Li, S. Yin, and Z. Liu, “Chromatic confocal microscopy using supercontinuum light,” Opt. Express 12, 2096–2101 (2004).
[Crossref] [PubMed]

Appl. Opt. (1)

Biophys. J. (1)

M. Schneideret al., “Two-Photon activation and excitation properties of PA-GFP in the 720–920-nm region,” Biophys. J. 89, 1346–1352 (2005).
[Crossref] [PubMed]

Biopolymers (1)

E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. 1. Conceptual basis and theory,” Biopolymers 13, 1–27 (1974).
[Crossref]

Chem. Phys. Lett. (1)

G. A. Blabet al., “Two-photon excitation action cross-sections of the autofluorescent proteins,” Chem. Phys. Lett. 350, 71–77 (2001).
[Crossref]

J. Microsc. (1)

K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223, 83–87 (2006).
[Crossref] [PubMed]

J. of Microsc. (1)

K. Konig, “Multiphoton microscopy in life sciences,” J. of Microsc. 200, 83–104 (2000).
[Crossref]

J. Opt. Soc. Am. B (3)

Nature (1)

D. Meshulach and Y. Silberberg, “Coherent quantum control of two-photon transitions by a femtosecond laser pulse,” Nature 396, 239–242 (1998).
[Crossref]

Opt. Express (2)

Opt. Lett. (3)

Proc. SPIE (1)

C. Tunget al., “Effects of index-mismatch-induced spherical aberration on two-photon imaging in skin and tissue-like constructs,” in Multiphoton Microscopy in the Biomedical Sciences III, A. Periasamy and P. T. C. So, eds., Proc. SPIE 4963, 95–104 (2003).
[Crossref]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “2-Photon Laser Scanning Fluorescence Microscopy,” Science 248, 73–76 (1990).
[Crossref] [PubMed]

Other (1)

G. P. Agrawal, Nonlinear Fiber Optics, 2nd ed. (Academic Press, London, 1995).

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Figures (4)

Fig. 1.
Fig. 1.

Schematic diagram of the experimental setup. L1: 150 mm focal length. L2: 300mm focal length. L3: objective lens, achromatic, N.A. 1.25.

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Fig. 2.
Fig. 2.

(a). Spectral broadening of femtosecond laser pulses using photonic crystal fiber. The broadened spectrum was generated by coupling femto-second laser pulses into a photonic crystal fiber (Blazephotonics SC-5.0-1040, 2 meters). The output was coupled into a multimode fiber and its spectrum was measured by an optical spectrum analyzer (Ando AQ 6315E). (b) A snapshot of two-photon fluorescence signal of rhodamine green excited at multiple excitation wavelengths (horizontal axis) simultaneously. The heterogeneity of the 2P-fluorescence image correlates with the excitation wavelength band shown in (a).

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Fig. 3.
Fig. 3.

Excitation-power dependence of the observed rhodamine fluorescence reveals genuine two-photon excitation. The slope of logarithmic dependence of fluorescence signal on the total excitation power for different wavelengths is ~1.95 (at the central region), and ~ 1.7 (near the spectral edge). ‘+’ shows the power dependence at excitation wavelength 818 nm. ‘o’ shows the power dependence at excitation wavelength 750 nm. ‘∗’ shows the power dependence at excitation wavelength 863 nm. The excitation power at a particular wavelength is proportional to the total excitation power. The inset shows the slope at different excitation wavelengths.

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Fig. 4.
Fig. 4.

(a). Relative measurements of two-photon action cross section of Rhodamine Green. Solid line: data measured by using single-shot line excitation method; ‘o’: data measured by slit-scanning method; ‘∗’: data measured by conventional approach using tunable laser. (b) Relative measurements of two-photon action cross section of DiI-C18. ‘o’: two-photon action cross section measured by single-shot line excitation method. ‘∗’: two-photon action cross section published in Ref. [3] by using tunable laser method.

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Equations (4)

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< F ( t ) > = 1 2 ϕ η C σ < I 2 ( t ) > ∫ V d V S 2 ( r ) ≈ 1 2 g ϕ η C σ 8 n < P ( t ) > 2 π λ
< F U ( t ) > < F R ( t ) > = ( ϕ U ϕ R ) ( C U C R ) ( n U n R ) ( η U σ U η R σ R ) = ( f U f R × d U d R ) ( C U C R ) ( n U n R ) ( η U σ U η R σ R )
σ TPE U = η U σ U = ( < F U ( t ) > < F R ( t ) > ) ( f R f U × d R d U ) ( C R C U ) σ TPE R
σ TPE U = ( < F U ( t ) > < F R ( t ) > ) ( C R C U ) σ TPE R

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