Abstract

We report polarization coherent anti-Stokes Raman scattering (P-CARS) microscopy that allows vibrational imaging with high sensitivity and spectral selectivity. The nonresonant background signals from both Raman scatterers and the solvent are efficiently suppressed in P-CARS microscopy. We demonstrate P-CARS imaging of unstained cells based on the contrast of the protein amide I band.

© 2001 Optical Society of America

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  1. M. D. Duncan, J. Reintjes, and T. J. Manuccia, Opt. Lett. 7, 350 (1982).
    [CrossRef] [PubMed]
  2. M. D. Duncan, J. Reintjes, and T. J. Manuccia, Opt. Eng. 24, 352 (1985).
  3. A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
    [CrossRef]
  4. M. Hashimoto, T. Araki, and S. Kawata, Opt. Lett. 25, 1768 (2000).
    [CrossRef]
  5. J. X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, J. Phys. Chem. B 105, 1277 (2001).
  6. A. Volkmer, J.-X. Cheng, and X. S. Xie, Phys. Rev. Lett. 87, 023901 (2001).
    [CrossRef]
  7. M. Müller, J. Squier, C. A. de Lange, and G. J. Brakenhoff, J. Microsc. (Oxford) 197, 150 (2000).
    [CrossRef]
  8. E. O. Potma, W. P. D. Boeij, and D. A. Wiersma, J. Opt. Soc. Am. B 17, 1678 (2000).
    [CrossRef]
  9. M. D. Levenson and S. S. Kano, Introduction to Nonlinear Laser Spectroscopy (Academic, San Diego, Calif., 1988), p. 148.
  10. S. Maeda, T. Kamisuki, and Y. Adachi, in Advances in Nonlinear Spectroscopy, R. J. H. Clark and R. E. Hester, eds. (Wiley, New York, 1988), pp. 253–297.
  11. S. A. Akhmanov, A. F. Bunkin, S. G. Ivanov, and N. I. Koroteev, JETP Lett. 25, 416 (1977).
  12. J.-L. Oudar, R. W. Smith, and Y. R. Shen, Appl. Phys. Lett. 34, 758 (1979).
    [CrossRef]
  13. R. Brakel and F. W. Schneider, in Advances in Nonlinear Spectroscopy, R. J. H. Clark and R. E. Hester, eds. (Wiley, New York, 1988), pp. 149–192.

2001 (2)

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

A. Volkmer, J.-X. Cheng, and X. S. Xie, Phys. Rev. Lett. 87, 023901 (2001).
[CrossRef]

2000 (3)

1999 (1)

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

1985 (1)

M. D. Duncan, J. Reintjes, and T. J. Manuccia, Opt. Eng. 24, 352 (1985).

1982 (1)

1979 (1)

J.-L. Oudar, R. W. Smith, and Y. R. Shen, Appl. Phys. Lett. 34, 758 (1979).
[CrossRef]

1977 (1)

S. A. Akhmanov, A. F. Bunkin, S. G. Ivanov, and N. I. Koroteev, JETP Lett. 25, 416 (1977).

Adachi, Y.

S. Maeda, T. Kamisuki, and Y. Adachi, in Advances in Nonlinear Spectroscopy, R. J. H. Clark and R. E. Hester, eds. (Wiley, New York, 1988), pp. 253–297.

Akhmanov, S. A.

S. A. Akhmanov, A. F. Bunkin, S. G. Ivanov, and N. I. Koroteev, JETP Lett. 25, 416 (1977).

Araki, T.

Boeij, W. P. D.

Book, L. D.

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

Brakel, R.

R. Brakel and F. W. Schneider, in Advances in Nonlinear Spectroscopy, R. J. H. Clark and R. E. Hester, eds. (Wiley, New York, 1988), pp. 149–192.

Brakenhoff, G. J.

M. Müller, J. Squier, C. A. de Lange, and G. J. Brakenhoff, J. Microsc. (Oxford) 197, 150 (2000).
[CrossRef]

Bunkin, A. F.

S. A. Akhmanov, A. F. Bunkin, S. G. Ivanov, and N. I. Koroteev, JETP Lett. 25, 416 (1977).

Cheng, J. X.

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

Cheng, J.-X.

A. Volkmer, J.-X. Cheng, and X. S. Xie, Phys. Rev. Lett. 87, 023901 (2001).
[CrossRef]

de Lange, C. A.

M. Müller, J. Squier, C. A. de Lange, and G. J. Brakenhoff, J. Microsc. (Oxford) 197, 150 (2000).
[CrossRef]

Duncan, M. D.

M. D. Duncan, J. Reintjes, and T. J. Manuccia, Opt. Eng. 24, 352 (1985).

M. D. Duncan, J. Reintjes, and T. J. Manuccia, Opt. Lett. 7, 350 (1982).
[CrossRef] [PubMed]

Hashimoto, M.

Holtom, G. R.

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

Ivanov, S. G.

S. A. Akhmanov, A. F. Bunkin, S. G. Ivanov, and N. I. Koroteev, JETP Lett. 25, 416 (1977).

Kamisuki, T.

S. Maeda, T. Kamisuki, and Y. Adachi, in Advances in Nonlinear Spectroscopy, R. J. H. Clark and R. E. Hester, eds. (Wiley, New York, 1988), pp. 253–297.

Kano, S. S.

M. D. Levenson and S. S. Kano, Introduction to Nonlinear Laser Spectroscopy (Academic, San Diego, Calif., 1988), p. 148.

Kawata, S.

Koroteev, N. I.

S. A. Akhmanov, A. F. Bunkin, S. G. Ivanov, and N. I. Koroteev, JETP Lett. 25, 416 (1977).

Levenson, M. D.

M. D. Levenson and S. S. Kano, Introduction to Nonlinear Laser Spectroscopy (Academic, San Diego, Calif., 1988), p. 148.

Maeda, S.

S. Maeda, T. Kamisuki, and Y. Adachi, in Advances in Nonlinear Spectroscopy, R. J. H. Clark and R. E. Hester, eds. (Wiley, New York, 1988), pp. 253–297.

Manuccia, T. J.

M. D. Duncan, J. Reintjes, and T. J. Manuccia, Opt. Eng. 24, 352 (1985).

M. D. Duncan, J. Reintjes, and T. J. Manuccia, Opt. Lett. 7, 350 (1982).
[CrossRef] [PubMed]

Müller, M.

M. Müller, J. Squier, C. A. de Lange, and G. J. Brakenhoff, J. Microsc. (Oxford) 197, 150 (2000).
[CrossRef]

Oudar, J.-L.

J.-L. Oudar, R. W. Smith, and Y. R. Shen, Appl. Phys. Lett. 34, 758 (1979).
[CrossRef]

Potma, E. O.

Reintjes, J.

M. D. Duncan, J. Reintjes, and T. J. Manuccia, Opt. Eng. 24, 352 (1985).

M. D. Duncan, J. Reintjes, and T. J. Manuccia, Opt. Lett. 7, 350 (1982).
[CrossRef] [PubMed]

Schneider, F. W.

R. Brakel and F. W. Schneider, in Advances in Nonlinear Spectroscopy, R. J. H. Clark and R. E. Hester, eds. (Wiley, New York, 1988), pp. 149–192.

Shen, Y. R.

J.-L. Oudar, R. W. Smith, and Y. R. Shen, Appl. Phys. Lett. 34, 758 (1979).
[CrossRef]

Smith, R. W.

J.-L. Oudar, R. W. Smith, and Y. R. Shen, Appl. Phys. Lett. 34, 758 (1979).
[CrossRef]

Squier, J.

M. Müller, J. Squier, C. A. de Lange, and G. J. Brakenhoff, J. Microsc. (Oxford) 197, 150 (2000).
[CrossRef]

Volkmer, A.

A. Volkmer, J.-X. Cheng, and X. S. Xie, Phys. Rev. Lett. 87, 023901 (2001).
[CrossRef]

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

Wiersma, D. A.

Xie, X. S.

A. Volkmer, J.-X. Cheng, and X. S. Xie, Phys. Rev. Lett. 87, 023901 (2001).
[CrossRef]

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

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

Zumbusch, A.

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

Appl. Phys. Lett. (1)

J.-L. Oudar, R. W. Smith, and Y. R. Shen, Appl. Phys. Lett. 34, 758 (1979).
[CrossRef]

J. Microsc. (Oxford) (1)

M. Müller, J. Squier, C. A. de Lange, and G. J. Brakenhoff, J. Microsc. (Oxford) 197, 150 (2000).
[CrossRef]

J. Opt. Soc. Am. B (1)

J. Phys. Chem. B (1)

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

JETP Lett. (1)

S. A. Akhmanov, A. F. Bunkin, S. G. Ivanov, and N. I. Koroteev, JETP Lett. 25, 416 (1977).

Opt. Eng. (1)

M. D. Duncan, J. Reintjes, and T. J. Manuccia, Opt. Eng. 24, 352 (1985).

Opt. Lett. (2)

Phys. Rev. Lett. (2)

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

A. Volkmer, J.-X. Cheng, and X. S. Xie, Phys. Rev. Lett. 87, 023901 (2001).
[CrossRef]

Other (3)

R. Brakel and F. W. Schneider, in Advances in Nonlinear Spectroscopy, R. J. H. Clark and R. E. Hester, eds. (Wiley, New York, 1988), pp. 149–192.

M. D. Levenson and S. S. Kano, Introduction to Nonlinear Laser Spectroscopy (Academic, San Diego, Calif., 1988), p. 148.

S. Maeda, T. Kamisuki, and Y. Adachi, in Advances in Nonlinear Spectroscopy, R. J. H. Clark and R. E. Hester, eds. (Wiley, New York, 1988), pp. 253–297.

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Figures (4)

Fig. 1
Fig. 1

(a) Polarization vectors of the pump and the Stokes fields, the nonresonant CARS signal, and the analyzer polarizer. (b) Nonresonant CARS signal [counts/s (CPS)] from a water–glass interface as a function of angle φ. The ratio of the maximum counts at φ30° to the minimum counts at φ120° is 600:1.

Fig. 2
Fig. 2

Schematic of the P-CARS microscope: P, polarizer; HW, half-wave plate; QW, quarter-wave plate; D, dichroic mirror. The lower objective is a water objective (N.A., 1.2; Olympus). The upper objective is an oil objective (N.A., 1.4; Nikon). The detector is an avalanche photodiode connected to a digital counter (SRS, SR620) and a homemade data-acquisition system.

Fig. 3
Fig. 3

(a) P-CARS spectrum of a 1μm polystyrene bead spin coated on a coverslip and covered with water, taken with a pump power of 350 μW and a Stokes power of 250 μW at a repetition rate of 50  kHz. The CARS spectrum of a polystyrene film was taken with a pump power of 780 μW and a Stokes power of 390 μW at a repetition rate of 100  kHz. The pump frequency was fixed at 13325 cm-1. The Stokes frequency was tuned from 11 691 to 11780 cm-1. The P-CARS signals are multiplied by a factor of 30. The spontaneous Raman spectrum of polystyrene was recorded on a Raman spectrometer (Jobin Yvon-Spex, LabRam). (b) CARS image of 1μm polystyrene beads spin coated on a coverslip and covered with water. The pump and Stokes powers were 0.6 and 0.3  mW, respectively, at a repetition rate of 400  kHz. (c)–(e) P-CARS images of the same polystyrene beads with ωPωS tuned to 1601, 1582, and 1553 cm-1. The pump and Stokes powers were 1.4 and 0.7  mW, respectively, at a repetition rate of 400  kHz. The acquisition time was 1.0  min for each image in (b)–(e). Shown below the images are the intensity profiles across the lines indicated by the arrows.

Fig. 4
Fig. 4

(a) CARS image of an epithelial cell without polarization-sensitive detection, ωPωS was tuned to the protein amide band. The pump and Stokes powers were 0.4 and 0.2  mW at a 400-kHz repetition rate. The acquisition time was 4  min. (b) P-CARS and Raman spectra of pure N-methylacetamide liquid. The P-CARS spectrum was recorded with average pump and Stokes powers of 1.6 and 0.8  mW at a pulse repetition rate of 400  kHz. (c) P-CARS image of an unstained epithelial cell with ωPωS tuned to 1650 cm-1. (d) Same as (c) but with ωPωS tuned to 1745 cm-1. The pump and Stokes powers were 1.8 and 1.0  mW, respectively, at a repetition rate of 400  kHz. The acquisition time was 8  min. Shown below the images are the intensity profiles across the lines indicated in the images.

Equations (5)

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PxNR=3χ1111NREP2ES*cosϕ,PyNR=3χ2112NREP2ES*sinϕ.
PxR=3χ1111REP2ES*cosϕ,PyR=3χ2112REP2ES*sinϕ.
PNR=3χ1111NREP2ES*cosϕ/cosα,
P=3EP2ES*χ1111Rcosϕsinα-ρRsinϕcosα.
rP2/PNR2=rχ1111R/2χ1111NR21-ρR/ρNR2 sin22α.

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