Abstract

We demonstrate a new approach to coherent anti-Stokes Raman scattering (CARS) microscopy that significantly increases the detection sensitivity. CARS signals are generated by collinearly overlapped, tightly focused, and raster scanned pump and Stokes laser beams, whose difference frequency is rapidly modulated. The resulting amplitude modulation of the CARS signal is detected through a lock-in amplifier. This scheme efficiently suppresses the nonresonant background and allows for the detection of far fewer vibrational oscillators than possible through existing CARS microscopy methods.

© 2006 Optical Society of America

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  1. J. X. Cheng and X. S. Xie, J. Phys. Chem. B 108, 827 (2004).
    [CrossRef]
  2. C. L. Evans, E. O. Potma, M. Pourgish'haag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. U.S.A. 102, 16807 (2005).
    [CrossRef] [PubMed]
  3. X. L. Nan, J. X. Cheng, and X. S. Xie, J. Lipid Res. 44, 2202 (2003).
    [CrossRef] [PubMed]
  4. N. Dudovich, D. Oron, and Y. Silberberg, Nature 418, 512 (2002).
    [CrossRef] [PubMed]
  5. D. Oron, N. Dudovich, and Y. Silberberg, Phys. Rev. Lett. 89, 273001 (2002).
    [CrossRef]
  6. E. O. Potma, C. L. Evans, and X. S. Xie, Opt. Lett. 31, 241 (2006).
    [CrossRef] [PubMed]
  7. E. Frumker, D. Oron, D. Mandelik, and Y. Silberberg, Opt. Lett. 29, 890 (2004).
    [CrossRef] [PubMed]
  8. L. Li, H. Wang, and J. X. Cheng, Biophys. J. 89, 3480 (2005).
    [CrossRef] [PubMed]
  9. M. Schwartz, A. Moradi-Araghi, and W. H. Koehler, J. Mol. Struct. 63, 279 (1980).
    [CrossRef]
  10. F. Ganikhanov, S. Carrasco, X. S. Xie, M. Katz, W. Steitz, and D. Kopf, Opt. Lett. 31, 1292 (2006).
    [CrossRef] [PubMed]

2006 (2)

2005 (2)

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

L. Li, H. Wang, and J. X. Cheng, Biophys. J. 89, 3480 (2005).
[CrossRef] [PubMed]

2004 (2)

2003 (1)

X. L. Nan, J. X. Cheng, and X. S. Xie, J. Lipid Res. 44, 2202 (2003).
[CrossRef] [PubMed]

2002 (2)

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

D. Oron, N. Dudovich, and Y. Silberberg, Phys. Rev. Lett. 89, 273001 (2002).
[CrossRef]

1980 (1)

M. Schwartz, A. Moradi-Araghi, and W. H. Koehler, J. Mol. Struct. 63, 279 (1980).
[CrossRef]

Carrasco, S.

Cheng, J. X.

L. Li, H. Wang, and J. X. Cheng, Biophys. J. 89, 3480 (2005).
[CrossRef] [PubMed]

J. X. Cheng and X. S. Xie, J. Phys. Chem. B 108, 827 (2004).
[CrossRef]

X. L. Nan, J. X. Cheng, and X. S. Xie, J. Lipid Res. 44, 2202 (2003).
[CrossRef] [PubMed]

Côté, D.

C. L. Evans, E. O. Potma, M. Pourgish'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]

D. Oron, N. Dudovich, and Y. Silberberg, Phys. Rev. Lett. 89, 273001 (2002).
[CrossRef]

Evans, C. L.

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

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

Frumker, E.

Ganikhanov, F.

Katz, M.

Koehler, W. H.

M. Schwartz, A. Moradi-Araghi, and W. H. Koehler, J. Mol. Struct. 63, 279 (1980).
[CrossRef]

Kopf, D.

Li, L.

L. Li, H. Wang, and J. X. Cheng, Biophys. J. 89, 3480 (2005).
[CrossRef] [PubMed]

Lin, C. P.

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

Mandelik, D.

Moradi-Araghi, A.

M. Schwartz, A. Moradi-Araghi, and W. H. Koehler, J. Mol. Struct. 63, 279 (1980).
[CrossRef]

Nan, X. L.

X. L. Nan, J. X. Cheng, and X. S. Xie, J. Lipid Res. 44, 2202 (2003).
[CrossRef] [PubMed]

Oron, D.

E. Frumker, D. Oron, D. Mandelik, and Y. Silberberg, Opt. Lett. 29, 890 (2004).
[CrossRef] [PubMed]

D. Oron, N. Dudovich, and Y. Silberberg, Phys. Rev. Lett. 89, 273001 (2002).
[CrossRef]

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. Pourgish'haag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. U.S.A. 102, 16807 (2005).
[CrossRef] [PubMed]

Pourgish'haag, M.

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

Schwartz, M.

M. Schwartz, A. Moradi-Araghi, and W. H. Koehler, J. Mol. Struct. 63, 279 (1980).
[CrossRef]

Silberberg, Y.

E. Frumker, D. Oron, D. Mandelik, and Y. Silberberg, Opt. Lett. 29, 890 (2004).
[CrossRef] [PubMed]

D. Oron, N. Dudovich, and Y. Silberberg, Phys. Rev. Lett. 89, 273001 (2002).
[CrossRef]

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

Steitz, W.

Wang, H.

L. Li, H. Wang, and J. X. Cheng, Biophys. J. 89, 3480 (2005).
[CrossRef] [PubMed]

Xie, X. S.

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

F. Ganikhanov, S. Carrasco, X. S. Xie, M. Katz, W. Steitz, and D. Kopf, Opt. Lett. 31, 1292 (2006).
[CrossRef] [PubMed]

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

J. X. Cheng and X. S. Xie, J. Phys. Chem. B 108, 827 (2004).
[CrossRef]

X. L. Nan, J. X. Cheng, and X. S. Xie, J. Lipid Res. 44, 2202 (2003).
[CrossRef] [PubMed]

Biophys. J. (1)

L. Li, H. Wang, and J. X. Cheng, Biophys. J. 89, 3480 (2005).
[CrossRef] [PubMed]

J. Lipid Res. (1)

X. L. Nan, J. X. Cheng, and X. S. Xie, J. Lipid Res. 44, 2202 (2003).
[CrossRef] [PubMed]

J. Mol. Struct. (1)

M. Schwartz, A. Moradi-Araghi, and W. H. Koehler, J. Mol. Struct. 63, 279 (1980).
[CrossRef]

J. Phys. Chem. B (1)

J. X. Cheng and X. S. Xie, J. Phys. Chem. B 108, 827 (2004).
[CrossRef]

Nature (1)

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

Opt. Lett. (3)

Phys. Rev. Lett. (1)

D. Oron, N. Dudovich, and Y. Silberberg, Phys. Rev. Lett. 89, 273001 (2002).
[CrossRef]

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

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

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Figures (3)

Fig. 1
Fig. 1

a, Components of the CARS signal. The last three terms in relation (1) are plotted versus detuning Δ = ω p ω s Ω R . Ω R is the center frequency of a homogeneously broadened Raman line with linewidth Γ. The curves are calculated with an assumption that χ NR ( 3 ) = 1.2 × χ R ( 3 ) ( Δ = 0 ) . b, Schematic of the FM CARS process. Solid curve, sum of the contributions from a. Dashed curve, nonresonant background. The resonance acts as an FM-to-AM converter, resulting in an amplitude-modulated signal that can be detected by using a lock-in amplifier. c, Schematic of the experimental setup. PC, Pockels cell; P, two-port Glan–Taylor prism; A, Glan–Thompson prism; DM, dichroic mirror, HWP, λ 2 plate. A circled cross and an arrow indicate Pump-1 and Pump-2 polarizations, respectively.

Fig. 2
Fig. 2

Forward CARS microscopy image, a, of 0.36 μ m diameter polystyrene beads taken at Δ = 3050 cm 1 . c, FM CARS image of the same area (Pump-1, 3050 cm 1 ; Pump-2, 3000 cm 1 ). b, d, Corresponding cross-sectional profiles of the images along the indicated line show the magnitude of nonresonant background suppression. e, Forward CARS image of a fixed A549 human lung cancer cell cultured with deuterium-labeled oleic acids taken at Δ = 2100 cm 1 . f, FM CARS image obtained when toggling between 2060 and 2100 cm 1 . Nonresonant background components have been significantly reduced by the FM CARS method.

Fig. 3
Fig. 3

FM CARS signal versus dilution factor, n. I is the FM CARS signal intensity from methanol dissolved in water, while I max is the CARS signal intensity from a pure methanol sample. Filled circles represent the experimental data points taken at a detector bandwidth of 25 kHz . Open circles correspond to data taken when the detector bandwidth was set to 1.6 Hz . The solid line is a plot of Eq. (2).

Equations (2)

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I CARS ( Ω ) χ R ( 3 ) ( Ω ) + χ NR ( 3 ) 2 = χ R ( 3 ) ( Ω ) 2 + ( χ NR ( 3 ) ) 2 + 2 Re { χ R ( 3 ) ( Ω ) } χ NR ( 3 ) .
I ( Δ , n ) = I H 2 O [ ( Γ 2 ) 2 Δ 2 + ( Γ 2 ) 2 ] [ R n 2 2 R ( 2 Δ Γ ) n ] ,

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