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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2006

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. Ogilvie, "Use of coherent control for selective two-photon fluorescence microscopy in live organisms," Opt. Express 14, 759-766 (2006).
[CrossRef] [PubMed]

2005

M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, "Two-Photon activation and excitation properties of PA-GFP in the 720-920-nm region," Biophys. J. 89, 1346-1352 (2005).
[CrossRef] [PubMed]

2004

2003

2001

G. A. Blab et al., "Two-photon excitation action cross-sections of the autofluorescent proteins," Chem. Phys. Lett. 350, 71-77 (2001).
[CrossRef]

2000

1999

1998

1996

1990

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

1974

E. L. Elson, and D. Magde, "Fluorescence correlation spectroscopy. 1. Conceptual basis and theory," Biopolymers 13, 1-27 (1974).
[CrossRef]

Albota, M. A.

Atkin, D. M.

Birks, T. A.

Blab, G. A.

G. A. Blab et 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

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.

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.

Biophys. J.

M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, "Two-Photon activation and excitation properties of PA-GFP in the 720-920-nm region," Biophys. J. 89, 1346-1352 (2005).
[CrossRef] [PubMed]

Biopolymers

E. L. Elson, and D. Magde, "Fluorescence correlation spectroscopy. 1. Conceptual basis and theory," Biopolymers 13, 1-27 (1974).
[CrossRef]

Chem. Phys. Lett.

G. A. Blab et al., "Two-photon excitation action cross-sections of the autofluorescent proteins," Chem. Phys. Lett. 350, 71-77 (2001).
[CrossRef]

J. Microsc.

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.

K. Konig, "Multiphoton microscopy in life sciences," J. of Microsc. 200, 83-104 (2000).
[CrossRef]

J. Opt. Soc. Am. B

Nature

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

Opt. Lett.

Science

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

Other

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

C. Tung et 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]

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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.

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).

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.

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.

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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