Abstract

We introduce dual-pump coherent anti-Stokes–Raman scattering (dual-CARS) microscopy. This new technique permits simultaneous imaging of two species characterized by different molecular vibrations, as well as the removal of nonresonant background. This is achieved by using three synchronized laser pulses probing two different vibrations. We demonstrate the virtues of the method by imaging a mixture of nondeuterated and deuterated lipids, clearly distinguishing the individual components and their organization in the mixed arrangement. Further, dual-CARS images of lipid stores in living Caenorhabditis elegans nematodes show that the suppression of the nonresonant background results in significantly enhanced image contrast.

© 2006 Optical Society of America

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  1. A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
    [CrossRef]
  2. C. L. Evans, E. O. Potma, M. Puoris'haag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. U.S.A. 102, 16807 (2005).
    [CrossRef] [PubMed]
  3. E. O. Potma, X. S. Xie, L. Muntean, J. Preusser, D. Jones, J. Ye, S. R. Leone, W. D. Hinsberg, and W. Schade, J. Phys. Chem. B 108, 1296 (2004).
    [CrossRef]
  4. G. W. H. Wurpel, J. M. Schins, and M. Müller, J. Phys. Chem. B 108, 3400 (2004).
    [CrossRef]
  5. Y. R. Shen, The Principles of Nonlinear Optics (Wiley, 1984).
  6. J.-X. Cheng, L. D. Book, and X. S. Xie, Opt. Lett. 26, 1341 (2001).
    [CrossRef]
  7. A. Volkmer, L. D. Book, and X. S. Xie, Appl. Phys. Lett. 80, 1505 (2002).
    [CrossRef]
  8. J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, J. Phys. Chem. B 105, 1277 (2001).
    [CrossRef]
  9. M. Müller and J. M. Schins, J. Phys. Chem. B 106, 3715 (2002).
    [CrossRef]
  10. N. Dudovich, D. Oron, and Y. Silberberg, Nature 418, 512 (2002).
    [CrossRef] [PubMed]
  11. S.-H. Lim, A. G. Caster, O. Nicolet, and S. R. Leone, J. Phys. Chem. B 110, 5196 (2006).
    [CrossRef] [PubMed]
  12. E. O. Potma, C. L. Evans, and X. S. Xie, Opt. Lett. 31, 241 (2006).
    [CrossRef] [PubMed]
  13. F. Ganikhanov, C. L. Evans, B. G. Saar, and X. S. Xie, Opt. Lett. 31, 1872 (2006).
    [CrossRef] [PubMed]
  14. R. P. Lucht, Opt. Lett. 12, 78 (1987).
    [CrossRef] [PubMed]
  15. T. Hellerer, A. M. K. Enejder, and A. Zumbusch, Appl. Phys. Lett. 85, 25 (2004).
    [CrossRef]

2006 (3)

2005 (1)

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. U.S.A. 102, 16807 (2005).
[CrossRef] [PubMed]

2004 (3)

E. O. Potma, X. S. Xie, L. Muntean, J. Preusser, D. Jones, J. Ye, S. R. Leone, W. D. Hinsberg, and W. Schade, J. Phys. Chem. B 108, 1296 (2004).
[CrossRef]

G. W. H. Wurpel, J. M. Schins, and M. Müller, J. Phys. Chem. B 108, 3400 (2004).
[CrossRef]

T. Hellerer, A. M. K. Enejder, and A. Zumbusch, Appl. Phys. Lett. 85, 25 (2004).
[CrossRef]

2002 (3)

M. Müller and J. M. Schins, J. Phys. Chem. B 106, 3715 (2002).
[CrossRef]

N. Dudovich, D. Oron, and Y. Silberberg, Nature 418, 512 (2002).
[CrossRef] [PubMed]

A. Volkmer, L. D. Book, and X. S. Xie, Appl. Phys. Lett. 80, 1505 (2002).
[CrossRef]

2001 (2)

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, J. Phys. Chem. B 105, 1277 (2001).
[CrossRef]

J.-X. Cheng, L. D. Book, and X. S. Xie, Opt. Lett. 26, 1341 (2001).
[CrossRef]

1999 (1)

A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

1987 (1)

Book, L. D.

A. Volkmer, L. D. Book, and X. S. Xie, Appl. Phys. Lett. 80, 1505 (2002).
[CrossRef]

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, J. Phys. Chem. B 105, 1277 (2001).
[CrossRef]

J.-X. Cheng, L. D. Book, and X. S. Xie, Opt. Lett. 26, 1341 (2001).
[CrossRef]

Caster, A. G.

S.-H. Lim, A. G. Caster, O. Nicolet, and S. R. Leone, J. Phys. Chem. B 110, 5196 (2006).
[CrossRef] [PubMed]

Cheng, J.-X.

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, J. Phys. Chem. B 105, 1277 (2001).
[CrossRef]

J.-X. Cheng, L. D. Book, and X. S. Xie, Opt. Lett. 26, 1341 (2001).
[CrossRef]

Côté, D.

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. U.S.A. 102, 16807 (2005).
[CrossRef] [PubMed]

Dudovich, N.

N. Dudovich, D. Oron, and Y. Silberberg, Nature 418, 512 (2002).
[CrossRef] [PubMed]

Enejder, A. M. K.

T. Hellerer, A. M. K. Enejder, and A. Zumbusch, Appl. Phys. Lett. 85, 25 (2004).
[CrossRef]

Evans, C. L.

Ganikhanov, F.

Hellerer, T.

T. Hellerer, A. M. K. Enejder, and A. Zumbusch, Appl. Phys. Lett. 85, 25 (2004).
[CrossRef]

Hinsberg, W. D.

E. O. Potma, X. S. Xie, L. Muntean, J. Preusser, D. Jones, J. Ye, S. R. Leone, W. D. Hinsberg, and W. Schade, J. Phys. Chem. B 108, 1296 (2004).
[CrossRef]

Holtom, G. R.

A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Jones, D.

E. O. Potma, X. S. Xie, L. Muntean, J. Preusser, D. Jones, J. Ye, S. R. Leone, W. D. Hinsberg, and W. Schade, J. Phys. Chem. B 108, 1296 (2004).
[CrossRef]

Leone, S. R.

S.-H. Lim, A. G. Caster, O. Nicolet, and S. R. Leone, J. Phys. Chem. B 110, 5196 (2006).
[CrossRef] [PubMed]

E. O. Potma, X. S. Xie, L. Muntean, J. Preusser, D. Jones, J. Ye, S. R. Leone, W. D. Hinsberg, and W. Schade, J. Phys. Chem. B 108, 1296 (2004).
[CrossRef]

Lim, S.-H.

S.-H. Lim, A. G. Caster, O. Nicolet, and S. R. Leone, J. Phys. Chem. B 110, 5196 (2006).
[CrossRef] [PubMed]

Lin, C. P.

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. U.S.A. 102, 16807 (2005).
[CrossRef] [PubMed]

Lucht, R. P.

Müller, M.

G. W. H. Wurpel, J. M. Schins, and M. Müller, J. Phys. Chem. B 108, 3400 (2004).
[CrossRef]

M. Müller and J. M. Schins, J. Phys. Chem. B 106, 3715 (2002).
[CrossRef]

Muntean, L.

E. O. Potma, X. S. Xie, L. Muntean, J. Preusser, D. Jones, J. Ye, S. R. Leone, W. D. Hinsberg, and W. Schade, J. Phys. Chem. B 108, 1296 (2004).
[CrossRef]

Nicolet, O.

S.-H. Lim, A. G. Caster, O. Nicolet, and S. R. Leone, J. Phys. Chem. B 110, 5196 (2006).
[CrossRef] [PubMed]

Oron, D.

N. Dudovich, D. Oron, and Y. Silberberg, Nature 418, 512 (2002).
[CrossRef] [PubMed]

Potma, E. O.

E. O. Potma, C. L. Evans, and X. S. Xie, Opt. Lett. 31, 241 (2006).
[CrossRef] [PubMed]

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. U.S.A. 102, 16807 (2005).
[CrossRef] [PubMed]

E. O. Potma, X. S. Xie, L. Muntean, J. Preusser, D. Jones, J. Ye, S. R. Leone, W. D. Hinsberg, and W. Schade, J. Phys. Chem. B 108, 1296 (2004).
[CrossRef]

Preusser, J.

E. O. Potma, X. S. Xie, L. Muntean, J. Preusser, D. Jones, J. Ye, S. R. Leone, W. D. Hinsberg, and W. Schade, J. Phys. Chem. B 108, 1296 (2004).
[CrossRef]

Puoris'haag, M.

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. U.S.A. 102, 16807 (2005).
[CrossRef] [PubMed]

Saar, B. G.

Schade, W.

E. O. Potma, X. S. Xie, L. Muntean, J. Preusser, D. Jones, J. Ye, S. R. Leone, W. D. Hinsberg, and W. Schade, J. Phys. Chem. B 108, 1296 (2004).
[CrossRef]

Schins, J. M.

G. W. H. Wurpel, J. M. Schins, and M. Müller, J. Phys. Chem. B 108, 3400 (2004).
[CrossRef]

M. Müller and J. M. Schins, J. Phys. Chem. B 106, 3715 (2002).
[CrossRef]

Shen, Y. R.

Y. R. Shen, The Principles of Nonlinear Optics (Wiley, 1984).

Silberberg, Y.

N. Dudovich, D. Oron, and Y. Silberberg, Nature 418, 512 (2002).
[CrossRef] [PubMed]

Volkmer, A.

A. Volkmer, L. D. Book, and X. S. Xie, Appl. Phys. Lett. 80, 1505 (2002).
[CrossRef]

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, J. Phys. Chem. B 105, 1277 (2001).
[CrossRef]

Wurpel, G. W. H.

G. W. H. Wurpel, J. M. Schins, and M. Müller, J. Phys. Chem. B 108, 3400 (2004).
[CrossRef]

Xie, X. S.

E. O. Potma, C. L. Evans, and X. S. Xie, Opt. Lett. 31, 241 (2006).
[CrossRef] [PubMed]

F. Ganikhanov, C. L. Evans, B. G. Saar, and X. S. Xie, Opt. Lett. 31, 1872 (2006).
[CrossRef] [PubMed]

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. U.S.A. 102, 16807 (2005).
[CrossRef] [PubMed]

E. O. Potma, X. S. Xie, L. Muntean, J. Preusser, D. Jones, J. Ye, S. R. Leone, W. D. Hinsberg, and W. Schade, J. Phys. Chem. B 108, 1296 (2004).
[CrossRef]

A. Volkmer, L. D. Book, and X. S. Xie, Appl. Phys. Lett. 80, 1505 (2002).
[CrossRef]

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, J. Phys. Chem. B 105, 1277 (2001).
[CrossRef]

J.-X. Cheng, L. D. Book, and X. S. Xie, Opt. Lett. 26, 1341 (2001).
[CrossRef]

A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Ye, J.

E. O. Potma, X. S. Xie, L. Muntean, J. Preusser, D. Jones, J. Ye, S. R. Leone, W. D. Hinsberg, and W. Schade, J. Phys. Chem. B 108, 1296 (2004).
[CrossRef]

Zumbusch, A.

T. Hellerer, A. M. K. Enejder, and A. Zumbusch, Appl. Phys. Lett. 85, 25 (2004).
[CrossRef]

A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Appl. Phys. Lett. (2)

A. Volkmer, L. D. Book, and X. S. Xie, Appl. Phys. Lett. 80, 1505 (2002).
[CrossRef]

T. Hellerer, A. M. K. Enejder, and A. Zumbusch, Appl. Phys. Lett. 85, 25 (2004).
[CrossRef]

J. Phys. Chem. B (5)

S.-H. Lim, A. G. Caster, O. Nicolet, and S. R. Leone, J. Phys. Chem. B 110, 5196 (2006).
[CrossRef] [PubMed]

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, J. Phys. Chem. B 105, 1277 (2001).
[CrossRef]

M. Müller and J. M. Schins, J. Phys. Chem. B 106, 3715 (2002).
[CrossRef]

E. O. Potma, X. S. Xie, L. Muntean, J. Preusser, D. Jones, J. Ye, S. R. Leone, W. D. Hinsberg, and W. Schade, J. Phys. Chem. B 108, 1296 (2004).
[CrossRef]

G. W. H. Wurpel, J. M. Schins, and M. Müller, J. Phys. Chem. B 108, 3400 (2004).
[CrossRef]

Nature (1)

N. Dudovich, D. Oron, and Y. Silberberg, Nature 418, 512 (2002).
[CrossRef] [PubMed]

Opt. Lett. (4)

Phys. Rev. Lett. (1)

A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Proc. Natl. Acad. Sci. U.S.A. (1)

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. U.S.A. 102, 16807 (2005).
[CrossRef] [PubMed]

Other (1)

Y. R. Shen, The Principles of Nonlinear Optics (Wiley, 1984).

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Figures (3)

Fig. 1
Fig. 1

Scheme of the experimental setup for dual-CARS microscopy. The three excitation pulses are generated by a Nd : V pump laser and two OPOs, overlapped in time and space, and coupled into a laser scanning microscope. An objective focuses the beams in the sample, and the emitted CARS signal is collected by an aspherical lens. A short-pass beam splitter separates the dual-CARS signal into its two components for detection by separate photomultiplier tubes. BS, beam splitter; DM, dichroic mirror; SP, short-pass beam splitter; R, retroreflector.

Fig. 2
Fig. 2

Dual-CARS microscopy images of a mixture of deuterated and nondeuterated tripalmitin. Simultaneous measurements were made with the lasers tuned to (a) the CH 2 vibration at 2845 cm 1 , and (b) the C–D vibration at 2115 cm 1 . Image (c) is the corresponding overlay image, where the red (dark) color represents the CH 2 vibration, and the yellow (bright) color represents the C–D vibration. The organization of the different crystals here is clearly visualized. The image dimensions are 100 × 100 μ m 2 , and the integration times were 60 s each.

Fig. 3
Fig. 3

Elimination of the nonresonant background by means of dual-CARS microscopy, exemplified by CARS images of a living C. elegans nematode. Image (a) was recorded at the symmetric CH 2 stretch vibration ( 2845 cm 1 ) and image (c) at the C–D vibration ( 2115 cm 1 ) . Image (c) visualizes the nonresonant background and has the same color scale as image (a). Image (e) is the pure resonant image calculated from Eq. (2), clearly visualizing the small and depleted lipid stores in the feeding deficient species. (b), (d), and (f) show normalized intensity profiles along the white lines in the corresponding images (a), (c), and (e). Profiles (b) and (f) show an improvement in the signal-to-background ratio by a factor of 3 for the processed image (e). The image dimensions are 50 μ m × 50 μ m , and the integration times were 20 s each.

Equations (2)

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I CARS ( Ω ) χ CARS ( 3 ) ( Ω ) 2 I p 2 I s = χ r ( 3 ) ( Ω ) + χ nr ( 3 ) 2 I p 2 I s ,
I CARS , resonant = I CARS ( ω 1 ) I CARS ( ω 2 ) I 1 2 = ϵ 1 χ r ( 3 ) 2 I p 1 2 I s ,

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