Abstract

We present spectral interferometric four-wave mixing (FWM) microscopy with a nearly shot-noise limited sensitivity and with the capability of separating FWM signals from fluorescence signals. We analyze the requirements for obtaining the shot-noise limited sensitivity and experimentally achieve the sensitivity that is only 4-dB lower than the shotnoise limit. Moreover, we show that only FWM signals can be extracted through the Fourier filtering even when the FWM spectrum is overlapped and overwhelmed by the fluorescence spectrum. We demonstrate simultaneous acquisition of FWM and two-photon excited fluorescence images of fluorescent monodispersed polystyrene microspheres.

© 2006 Optical Society of America

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  1. R. Hellwarth and P. Christensen, "Nonlinear optical microscopic examination of structure in polycrystalline ZnSe," Opt. Commun. 12, 318-322 (1974).
    [CrossRef]
  2. J. N. Gannaway and C. J. R. Sheppard, "Second-harmonic imaging in the scanning optical microscope," Opt. and Quant. Elec. 10, 435-439 (1978).
    [CrossRef]
  3. Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-924 (1997).
    [CrossRef]
  4. M. Müller, J. Squier, K. R. Wilson, and G. J. Brankenhoff, "3D microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998).
    [CrossRef] [PubMed]
  5. M. D. Duncan, J. Reintjes, and T. J. Manuccia, "Scanning coherent anti-Stokes Raman microscope," Opt. Lett. 7, 350-352 (1982).
    [CrossRef] [PubMed]
  6. A. Zumbusch, G. R. Holtom and X. S. Xie, "Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering," Phys. Rev. Lett. 82, 4142-4145 (1999).
    [CrossRef]
  7. K. Isobe, S. Kataoka, R. Murase, W. Watanabe, T. Higashi, S. Kawakami, S. Matsunaga, K. Fukui, and K. Itoh, "Stimulated parametric emission microscopy," Opt. Express 14, 786-793 (2006).
    [CrossRef]
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    [CrossRef]
  9. A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Natl. Acad. Sci. USA 99, 11014-11019 (2002).
    [CrossRef] [PubMed]
  10. M. Kobayashi, K. Fujita, O. Nakamura, and S. Kawata, "Time-gated imaging for multifocus second-harmonic generation microscopy," Rev. Sci. Instrum. 76, 073704-1-073704-4 (2005).
    [CrossRef]
  11. R. K. Chang, J. Ducuing, and N. Bloembergen, "Relative phase measurement between fundamental and second-harmonic light," Phys. Rev. Lett. 15, 6-9 (1965).
    [CrossRef]
  12. G. Marowsky, and G. Lüpke, "CARS-background suppression by phase-controlled nonlinear interferometry," Appl. Phys. B 51, 49-51 (1990).
    [CrossRef]
  13. Y. Jiang, I. Tomov, Y. Wang, and Z. Chen, "Second-harmonic optical coherence tomography," Opt. Lett. 29, 1090-1092 (2004).
    [CrossRef] [PubMed]
  14. C. Vinegoni, J. S. Bredfeldt, and D. L. Marks, "Nonlinear optical contrast enhancement for optical coherence tomography," Opt. Express 12, 331-341 (2004).
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    [CrossRef] [PubMed]
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    [CrossRef]
  21. J. F. de Boer, B. Cense, B. H. Park, M. C. Pierce, G. J. Tearney, and B. E. Bouma, "Improved signal-to-noise ratio in spectral-domain compared with time-domain optical coherence tomography," Opt. Lett. 28, 2067-2069 (2003).
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    [CrossRef]
  24. P. T. So, C. Y. Dong, B. R. Masters and K. M. Berland, "Two-photon excitation fluorescence microscopy," Annu. Rev. Biomed. Eng. 2, 399-429 (2000).
    [CrossRef]

2006 (2)

2005 (1)

2004 (5)

2003 (2)

2002 (1)

A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Natl. Acad. Sci. USA 99, 11014-11019 (2002).
[CrossRef] [PubMed]

2000 (2)

L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, "Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy," Opt. Lett. 25, 320-322 (2000).
[CrossRef]

P. T. So, C. Y. Dong, B. R. Masters and K. M. Berland, "Two-photon excitation fluorescence microscopy," Annu. Rev. Biomed. Eng. 2, 399-429 (2000).
[CrossRef]

1999 (1)

A. Zumbusch, G. R. Holtom and X. S. Xie, "Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering," Phys. Rev. Lett. 82, 4142-4145 (1999).
[CrossRef]

1998 (1)

M. Müller, J. Squier, K. R. Wilson, and G. J. Brankenhoff, "3D microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998).
[CrossRef] [PubMed]

1997 (1)

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-924 (1997).
[CrossRef]

1990 (1)

G. Marowsky, and G. Lüpke, "CARS-background suppression by phase-controlled nonlinear interferometry," Appl. Phys. B 51, 49-51 (1990).
[CrossRef]

1989 (1)

R. Adair, L. L. Chase, and S. A. Payne, "Nonlinear refractive index of optical crystal," Phys. Rev. B 39, 3337-3350 (1989).
[CrossRef]

1982 (1)

1978 (1)

J. N. Gannaway and C. J. R. Sheppard, "Second-harmonic imaging in the scanning optical microscope," Opt. and Quant. Elec. 10, 435-439 (1978).
[CrossRef]

1974 (1)

R. Hellwarth and P. Christensen, "Nonlinear optical microscopic examination of structure in polycrystalline ZnSe," Opt. Commun. 12, 318-322 (1974).
[CrossRef]

1965 (1)

R. K. Chang, J. Ducuing, and N. Bloembergen, "Relative phase measurement between fundamental and second-harmonic light," Phys. Rev. Lett. 15, 6-9 (1965).
[CrossRef]

Adair, R.

R. Adair, L. L. Chase, and S. A. Payne, "Nonlinear refractive index of optical crystal," Phys. Rev. B 39, 3337-3350 (1989).
[CrossRef]

Applegate, B. E.

Barad, Y.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-924 (1997).
[CrossRef]

Berland, K. M.

P. T. So, C. Y. Dong, B. R. Masters and K. M. Berland, "Two-photon excitation fluorescence microscopy," Annu. Rev. Biomed. Eng. 2, 399-429 (2000).
[CrossRef]

Blanchard-Desce, M.

Bloembergen, N.

R. K. Chang, J. Ducuing, and N. Bloembergen, "Relative phase measurement between fundamental and second-harmonic light," Phys. Rev. Lett. 15, 6-9 (1965).
[CrossRef]

Bouma, B. E.

Brankenhoff, G. J.

M. Müller, J. Squier, K. R. Wilson, and G. J. Brankenhoff, "3D microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998).
[CrossRef] [PubMed]

Bredfeldt, J. S.

Cense, B.

Chang, R. K.

R. K. Chang, J. Ducuing, and N. Bloembergen, "Relative phase measurement between fundamental and second-harmonic light," Phys. Rev. Lett. 15, 6-9 (1965).
[CrossRef]

Chase, L. L.

R. Adair, L. L. Chase, and S. A. Payne, "Nonlinear refractive index of optical crystal," Phys. Rev. B 39, 3337-3350 (1989).
[CrossRef]

Chen, Z.

Christensen, P.

R. Hellwarth and P. Christensen, "Nonlinear optical microscopic examination of structure in polycrystalline ZnSe," Opt. Commun. 12, 318-322 (1974).
[CrossRef]

de Boer, J. F.

Dong, C. Y.

P. T. So, C. Y. Dong, B. R. Masters and K. M. Berland, "Two-photon excitation fluorescence microscopy," Annu. Rev. Biomed. Eng. 2, 399-429 (2000).
[CrossRef]

Ducuing, J.

R. K. Chang, J. Ducuing, and N. Bloembergen, "Relative phase measurement between fundamental and second-harmonic light," Phys. Rev. Lett. 15, 6-9 (1965).
[CrossRef]

Duncan, M. D.

Eisenberg, H.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-924 (1997).
[CrossRef]

Evans, C. L.

Ferchar, A. F.

Fukui, K.

Gannaway, J. N.

J. N. Gannaway and C. J. R. Sheppard, "Second-harmonic imaging in the scanning optical microscope," Opt. and Quant. Elec. 10, 435-439 (1978).
[CrossRef]

Hellwarth, R.

R. Hellwarth and P. Christensen, "Nonlinear optical microscopic examination of structure in polycrystalline ZnSe," Opt. Commun. 12, 318-322 (1974).
[CrossRef]

Higashi, T.

Hitzenberger, C. K.

Holtom, G. R.

A. Zumbusch, G. R. Holtom and X. S. Xie, "Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering," Phys. Rev. Lett. 82, 4142-4145 (1999).
[CrossRef]

Horowitz, M.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-924 (1997).
[CrossRef]

Isobe, K.

Itoh, K.

Izatt, J. A.

Jiang, Y.

Kataoka, S.

Kawakami, S.

Laiho, L. H.

Leitgeb, R.

Lüpke, G.

G. Marowsky, and G. Lüpke, "CARS-background suppression by phase-controlled nonlinear interferometry," Appl. Phys. B 51, 49-51 (1990).
[CrossRef]

Manuccia, T. J.

Marks, D. L.

Marowsky, G.

G. Marowsky, and G. Lüpke, "CARS-background suppression by phase-controlled nonlinear interferometry," Appl. Phys. B 51, 49-51 (1990).
[CrossRef]

Masters, B. R.

P. T. So, C. Y. Dong, B. R. Masters and K. M. Berland, "Two-photon excitation fluorescence microscopy," Annu. Rev. Biomed. Eng. 2, 399-429 (2000).
[CrossRef]

Matsunaga, S.

Mertz, J.

Moreaux, L.

Müller, M.

G. W. H. Wurpel, J. M. Schins and M. Müller, "Direct measurement of chain order in single lipid mono- and bilayers with multiplex CARS," J. Phys. Chem. B 108, 3400-3403 (2004).
[CrossRef]

M. Müller, J. Squier, K. R. Wilson, and G. J. Brankenhoff, "3D microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998).
[CrossRef] [PubMed]

Murase, R.

Park, B. H.

Payne, S. A.

R. Adair, L. L. Chase, and S. A. Payne, "Nonlinear refractive index of optical crystal," Phys. Rev. B 39, 3337-3350 (1989).
[CrossRef]

Pierce, M. C.

Potma, E. O.

Reintjes, J.

Sandre, O.

Sarunic, M. V.

Schins, J. M.

G. W. H. Wurpel, J. M. Schins and M. Müller, "Direct measurement of chain order in single lipid mono- and bilayers with multiplex CARS," J. Phys. Chem. B 108, 3400-3403 (2004).
[CrossRef]

Sheppard, C. J. R.

J. N. Gannaway and C. J. R. Sheppard, "Second-harmonic imaging in the scanning optical microscope," Opt. and Quant. Elec. 10, 435-439 (1978).
[CrossRef]

Silberberg, Y.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-924 (1997).
[CrossRef]

So, P. T.

P. T. So, C. Y. Dong, B. R. Masters and K. M. Berland, "Two-photon excitation fluorescence microscopy," Annu. Rev. Biomed. Eng. 2, 399-429 (2000).
[CrossRef]

So, P. T. C.

Squier, J.

M. Müller, J. Squier, K. R. Wilson, and G. J. Brankenhoff, "3D microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998).
[CrossRef] [PubMed]

Tearney, G. J.

Tomov, I.

Tromberg, B. J.

A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Natl. Acad. Sci. USA 99, 11014-11019 (2002).
[CrossRef] [PubMed]

Vinegoni, C.

Wang, Y.

Watanabe, W.

Wilson, K. R.

M. Müller, J. Squier, K. R. Wilson, and G. J. Brankenhoff, "3D microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998).
[CrossRef] [PubMed]

Wurpel, G. W. H.

G. W. H. Wurpel, J. M. Schins and M. Müller, "Direct measurement of chain order in single lipid mono- and bilayers with multiplex CARS," J. Phys. Chem. B 108, 3400-3403 (2004).
[CrossRef]

Xie, X. S.

Yazdanfar, S.

Yeh, A.

A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Natl. Acad. Sci. USA 99, 11014-11019 (2002).
[CrossRef] [PubMed]

Zoumi, A.

A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Natl. Acad. Sci. USA 99, 11014-11019 (2002).
[CrossRef] [PubMed]

Zumbusch, A.

A. Zumbusch, G. R. Holtom and X. S. Xie, "Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering," Phys. Rev. Lett. 82, 4142-4145 (1999).
[CrossRef]

Annu. Rev. Biomed. Eng. (1)

P. T. So, C. Y. Dong, B. R. Masters and K. M. Berland, "Two-photon excitation fluorescence microscopy," Annu. Rev. Biomed. Eng. 2, 399-429 (2000).
[CrossRef]

Appl. Phys. B (1)

G. Marowsky, and G. Lüpke, "CARS-background suppression by phase-controlled nonlinear interferometry," Appl. Phys. B 51, 49-51 (1990).
[CrossRef]

Appl. Phys. Lett. (1)

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-924 (1997).
[CrossRef]

J. Microsc. (1)

M. Müller, J. Squier, K. R. Wilson, and G. J. Brankenhoff, "3D microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998).
[CrossRef] [PubMed]

J. Phys. Chem. B (1)

G. W. H. Wurpel, J. M. Schins and M. Müller, "Direct measurement of chain order in single lipid mono- and bilayers with multiplex CARS," J. Phys. Chem. B 108, 3400-3403 (2004).
[CrossRef]

Opt. and Quant. Elec. (1)

J. N. Gannaway and C. J. R. Sheppard, "Second-harmonic imaging in the scanning optical microscope," Opt. and Quant. Elec. 10, 435-439 (1978).
[CrossRef]

Opt. Commun. (1)

R. Hellwarth and P. Christensen, "Nonlinear optical microscopic examination of structure in polycrystalline ZnSe," Opt. Commun. 12, 318-322 (1974).
[CrossRef]

Opt. Express (4)

Opt. Lett. (7)

Phys. Rev. B (1)

R. Adair, L. L. Chase, and S. A. Payne, "Nonlinear refractive index of optical crystal," Phys. Rev. B 39, 3337-3350 (1989).
[CrossRef]

Phys. Rev. Lett. (2)

R. K. Chang, J. Ducuing, and N. Bloembergen, "Relative phase measurement between fundamental and second-harmonic light," Phys. Rev. Lett. 15, 6-9 (1965).
[CrossRef]

A. Zumbusch, G. R. Holtom and X. S. Xie, "Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering," Phys. Rev. Lett. 82, 4142-4145 (1999).
[CrossRef]

Proc. Natl. Acad. Sci. USA (1)

A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Natl. Acad. Sci. USA 99, 11014-11019 (2002).
[CrossRef] [PubMed]

Other (2)

M. Kobayashi, K. Fujita, O. Nakamura, and S. Kawata, "Time-gated imaging for multifocus second-harmonic generation microscopy," Rev. Sci. Instrum. 76, 073704-1-073704-4 (2005).
[CrossRef]

K. Isobe, W. Watanabe, and K. Itoh, "Interferometric second-harmonic-generation microscopy," in Optics Japan 2002 Extended Abstracts (Optical Society of Japan (Japanese Society of Applied Physics), Tokyo, 2002), pp. 18-19.

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Figures (8)

Fig. 1.
Fig. 1.

Schematic diagram of a SI-FWM microscope.

Fig. 2.
Fig. 2.

Experimental setup of SI-FWM microscopy. CM: chirp mirror, BS: beam splitter, OB: objective lens, SPF: short-pass filter, CCD: charge coupled device.

Fig. 3.
Fig. 3.

Emission spectra and spectral interferogram. (a) Emission spectrum from a dye (Coumarin 120, 6 mM) dissolved in ethanol. (b) Emission spectrum obtained from a slide glass. (c) Spectral interferogram of (a) and (b).

Fig. 4.
Fig. 4.

Dependences of FWM (triangle) and TPEF (square) signal intensities I signal on incident intensity I inc.

Fig. 5.
Fig. 5.

Dependences of interferometric signal P signal (a) and SNR (b) on the LO power P LO.

Fig. 6.
Fig. 6.

One-dimensional distribution of FWM signal near the interface between the slide glass and air. The traces were measured with (a) heterodyne spectral interferometric detection and (b) direct detection.

Fig. 7.
Fig. 7.

Emission spectra from the mixed dye solution (Coumarin 120 and Coumarin 153). (a) Emission spectrum in the direct detection. (b) Spectral interferogram. (c) Reconstructed FWM spectrum.

Fig. 8.
Fig. 8.

Simultaneously obtained Images of fluorescent microbeads. (a) Interferometric FWM image. (b) TPEF image. Scale bar indicates 5 µm.

Equations (11)

Equations on this page are rendered with MathJax. Learn more.

K ( ω ) = η ρ Δ t 2 h ν [ E S ( ω ) 2 + E LO ( ω ) 2 + 2 E S ( ω ) E LO ( ω ) cos ( ω τ + ϕ S ϕ LO + ϕ 0 ) ] ,
S S ( ω ) d ω = η ρ Δ t 2 h ν P S P LO ,
K shot 2 ( ω ) = K ( ω ) ~ η ρ Δ t 2 h ν E LO ( ω ) 2 ,
N 2 N ( ω ) d ω 2 = 2 π K ˜ shot ( τ ) 2 = K shot ( ω ) e i ω t d ω 2
= K shot 2 ( ω ) d ω = K shot 2 ( ω ) d ω = η ρ Δ t 2 h ν P LO ,
N A 2 = η ρ Δ t 2 h ν P LO .
SNR shot = S 2 N A 2 = η ρ Δ t P S h ν .
K shot 2 ( ω ) Δ ω = K ( ω ) Δ ω ~ η ρ Δ t 2 h ν E LO ( ω ) 2 Δ ω .
Δ t 2 h ν ( C thermal 2 + C read 2 ) η ρ E LO ( ω ) 2 Δ ω .
η ρ E LO ( ω ) 2 Δ ω 2 h ν i dark q ,
Δ t < 2 h ν C max η ρ E LO ( ω ) 2 Δ ω .

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