Abstract

Coherent Anti-Stokes Raman Spectroscopy (CARS) is a non-linear process in which the energy difference of a pair of incoming photons matches the energy of the vibrational mode of a molecular bond of interest. This phonon population is coherently probed by a third photon and anti-Stokes radiation is emitted. Here a novel approach to CARS microscopy is presented yielding the intensity of the anti-Stokes emission, the directionality the molecular bonds of interest, and their average orientation. Myelinated axons in fixed mouse-brain slices have been imaged by RP-CARS. We were able to detect the local average direction of the acylic chains of membrane phospholipids and their spatial anisotropy. This novel method may impact the study of healthy brain circuitry as well as demyelinating diseases or other pathological states associated with altered neural connectivity.

© 2012 OSA

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  1. P. Maker and R. Terhune, “Study of optical effects due to an induced polarization third order in the electric field strength,” Phys. Rev.137(3A), A801–A818 (1965).
    [CrossRef]
  2. J. X. Cheng and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications,” J. Phys. Chem. B108(3), 827–840 (2004).
    [CrossRef]
  3. C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.102(46), 16807–16812 (2005).
    [CrossRef] [PubMed]
  4. C. L. Evans and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu Rev Anal Chem (Palo Alto Calif)1(1), 883–909 (2008).
    [CrossRef] [PubMed]
  5. J. X. Cheng, S. Pautot, D. A. Weitz, and X. S. Xie, “Ordering of water molecules between phospholipid bilayers visualized by coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.100(17), 9826–9830 (2003).
    [CrossRef] [PubMed]
  6. A. V. Kachynski, A. N. Kuzmin, P. N. Prasad, and I. I. Smalyukh, “Coherent anti-Stokes Raman scattering polarized microscopy of three-dimensional director structures in liquid crystals,” Appl. Phys. Lett.91(15), 151905 (2007).
    [CrossRef]
  7. F. Munhoz, H. Rigneault, and S. Brasselet, “Polarization-resolved four-wave mixing microscopy for structural imaging in thick tissues,” J. Opt. Soc. Am. B29(6), 1541–1550 (2012).
    [CrossRef]
  8. J. X. Cheng, L. D. Book, and X. S. Xie, “Polarization coherent anti-Stokes Raman scattering microscopy,” Opt. Lett.26(17), 1341–1343 (2001).
    [CrossRef] [PubMed]
  9. A. Volkmer, L. D. Book, and X. S. Xie, “Time-resolved coherent anti-Stokes Raman scattering microscopy: Imaging based on Raman free induction decay,” Appl. Phys. Lett.80(9), 1505–1507 (2002).
    [CrossRef]
  10. Y. Fu, T. B. Huff, H.-W. Wang, J.-X. Cheng, and H. Wang, “Ex vivo and in vivo imaging of myelin fibers in mouse brain by coherent anti-Stokes Raman scattering microscopy,” Opt. Express16(24), 19396–19409 (2008).
    [CrossRef] [PubMed]
  11. E. Bélanger, S. Bégin, S. Laffray, Y. De Koninck, R. Vallée, and D. Côté, “Quantitative myelin imaging with coherent anti-Stokes Raman scattering microscopy: alleviating the excitation polarization dependence with circularly polarized laser beams,” Opt. Express17(21), 18419–18432 (2009).
    [CrossRef] [PubMed]
  12. H. Wang, Y. Fu, P. Zickmund, R. Shi, and J. X. Cheng, “Coherent anti-Stokes Raman scattering imaging of axonal myelin in live spinal tissues,” Biophys. J.89(1), 581–591 (2005).
    [CrossRef] [PubMed]
  13. C. Shang and H. Hsu, “The spatial symmetric forms of third-order nonlinear susceptibility,” IEEE J. Quantum Electron.23(2), 177–179 (1987).
    [CrossRef]
  14. R. W. Hellwarth, “Third-order optical susceptibilities of liquids and solids,” Prog. Quantum Electron.5, 1–68 (1979).
    [CrossRef]
  15. M. Axer, K. Amunts, D. Grässel, C. Palm, J. Dammers, H. Axer, U. Pietrzyk, and K. Zilles, “A novel approach to the human connectome: ultra-high resolution mapping of fiber tracts in the brain,” Neuroimage54(2), 1091–1101 (2011).
    [CrossRef] [PubMed]
  16. M. D. Budde and J. A. Frank, “Examining brain microstructure using structure tensor analysis of histological sections,” Neuroimage63(1), 1–10 (2012).
    [CrossRef] [PubMed]

2012

M. D. Budde and J. A. Frank, “Examining brain microstructure using structure tensor analysis of histological sections,” Neuroimage63(1), 1–10 (2012).
[CrossRef] [PubMed]

F. Munhoz, H. Rigneault, and S. Brasselet, “Polarization-resolved four-wave mixing microscopy for structural imaging in thick tissues,” J. Opt. Soc. Am. B29(6), 1541–1550 (2012).
[CrossRef]

2011

M. Axer, K. Amunts, D. Grässel, C. Palm, J. Dammers, H. Axer, U. Pietrzyk, and K. Zilles, “A novel approach to the human connectome: ultra-high resolution mapping of fiber tracts in the brain,” Neuroimage54(2), 1091–1101 (2011).
[CrossRef] [PubMed]

2009

2008

Y. Fu, T. B. Huff, H.-W. Wang, J.-X. Cheng, and H. Wang, “Ex vivo and in vivo imaging of myelin fibers in mouse brain by coherent anti-Stokes Raman scattering microscopy,” Opt. Express16(24), 19396–19409 (2008).
[CrossRef] [PubMed]

C. L. Evans and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu Rev Anal Chem (Palo Alto Calif)1(1), 883–909 (2008).
[CrossRef] [PubMed]

2007

A. V. Kachynski, A. N. Kuzmin, P. N. Prasad, and I. I. Smalyukh, “Coherent anti-Stokes Raman scattering polarized microscopy of three-dimensional director structures in liquid crystals,” Appl. Phys. Lett.91(15), 151905 (2007).
[CrossRef]

2005

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J. X. Cheng, “Coherent anti-Stokes Raman scattering imaging of axonal myelin in live spinal tissues,” Biophys. J.89(1), 581–591 (2005).
[CrossRef] [PubMed]

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.102(46), 16807–16812 (2005).
[CrossRef] [PubMed]

2004

J. X. Cheng and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications,” J. Phys. Chem. B108(3), 827–840 (2004).
[CrossRef]

2003

J. X. Cheng, S. Pautot, D. A. Weitz, and X. S. Xie, “Ordering of water molecules between phospholipid bilayers visualized by coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.100(17), 9826–9830 (2003).
[CrossRef] [PubMed]

2002

A. Volkmer, L. D. Book, and X. S. Xie, “Time-resolved coherent anti-Stokes Raman scattering microscopy: Imaging based on Raman free induction decay,” Appl. Phys. Lett.80(9), 1505–1507 (2002).
[CrossRef]

2001

1987

C. Shang and H. Hsu, “The spatial symmetric forms of third-order nonlinear susceptibility,” IEEE J. Quantum Electron.23(2), 177–179 (1987).
[CrossRef]

1979

R. W. Hellwarth, “Third-order optical susceptibilities of liquids and solids,” Prog. Quantum Electron.5, 1–68 (1979).
[CrossRef]

1965

P. Maker and R. Terhune, “Study of optical effects due to an induced polarization third order in the electric field strength,” Phys. Rev.137(3A), A801–A818 (1965).
[CrossRef]

Amunts, K.

M. Axer, K. Amunts, D. Grässel, C. Palm, J. Dammers, H. Axer, U. Pietrzyk, and K. Zilles, “A novel approach to the human connectome: ultra-high resolution mapping of fiber tracts in the brain,” Neuroimage54(2), 1091–1101 (2011).
[CrossRef] [PubMed]

Axer, H.

M. Axer, K. Amunts, D. Grässel, C. Palm, J. Dammers, H. Axer, U. Pietrzyk, and K. Zilles, “A novel approach to the human connectome: ultra-high resolution mapping of fiber tracts in the brain,” Neuroimage54(2), 1091–1101 (2011).
[CrossRef] [PubMed]

Axer, M.

M. Axer, K. Amunts, D. Grässel, C. Palm, J. Dammers, H. Axer, U. Pietrzyk, and K. Zilles, “A novel approach to the human connectome: ultra-high resolution mapping of fiber tracts in the brain,” Neuroimage54(2), 1091–1101 (2011).
[CrossRef] [PubMed]

Bégin, S.

Bélanger, E.

Book, L. D.

A. Volkmer, L. D. Book, and X. S. Xie, “Time-resolved coherent anti-Stokes Raman scattering microscopy: Imaging based on Raman free induction decay,” Appl. Phys. Lett.80(9), 1505–1507 (2002).
[CrossRef]

J. X. Cheng, L. D. Book, and X. S. Xie, “Polarization coherent anti-Stokes Raman scattering microscopy,” Opt. Lett.26(17), 1341–1343 (2001).
[CrossRef] [PubMed]

Brasselet, S.

Budde, M. D.

M. D. Budde and J. A. Frank, “Examining brain microstructure using structure tensor analysis of histological sections,” Neuroimage63(1), 1–10 (2012).
[CrossRef] [PubMed]

Cheng, J. X.

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J. X. Cheng, “Coherent anti-Stokes Raman scattering imaging of axonal myelin in live spinal tissues,” Biophys. J.89(1), 581–591 (2005).
[CrossRef] [PubMed]

J. X. Cheng and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications,” J. Phys. Chem. B108(3), 827–840 (2004).
[CrossRef]

J. X. Cheng, S. Pautot, D. A. Weitz, and X. S. Xie, “Ordering of water molecules between phospholipid bilayers visualized by coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.100(17), 9826–9830 (2003).
[CrossRef] [PubMed]

J. X. Cheng, L. D. Book, and X. S. Xie, “Polarization coherent anti-Stokes Raman scattering microscopy,” Opt. Lett.26(17), 1341–1343 (2001).
[CrossRef] [PubMed]

Cheng, J.-X.

Côté, D.

E. Bélanger, S. Bégin, S. Laffray, Y. De Koninck, R. Vallée, and D. Côté, “Quantitative myelin imaging with coherent anti-Stokes Raman scattering microscopy: alleviating the excitation polarization dependence with circularly polarized laser beams,” Opt. Express17(21), 18419–18432 (2009).
[CrossRef] [PubMed]

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.102(46), 16807–16812 (2005).
[CrossRef] [PubMed]

Dammers, J.

M. Axer, K. Amunts, D. Grässel, C. Palm, J. Dammers, H. Axer, U. Pietrzyk, and K. Zilles, “A novel approach to the human connectome: ultra-high resolution mapping of fiber tracts in the brain,” Neuroimage54(2), 1091–1101 (2011).
[CrossRef] [PubMed]

De Koninck, Y.

Evans, C. L.

C. L. Evans and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu Rev Anal Chem (Palo Alto Calif)1(1), 883–909 (2008).
[CrossRef] [PubMed]

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.102(46), 16807–16812 (2005).
[CrossRef] [PubMed]

Frank, J. A.

M. D. Budde and J. A. Frank, “Examining brain microstructure using structure tensor analysis of histological sections,” Neuroimage63(1), 1–10 (2012).
[CrossRef] [PubMed]

Fu, Y.

Y. Fu, T. B. Huff, H.-W. Wang, J.-X. Cheng, and H. Wang, “Ex vivo and in vivo imaging of myelin fibers in mouse brain by coherent anti-Stokes Raman scattering microscopy,” Opt. Express16(24), 19396–19409 (2008).
[CrossRef] [PubMed]

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J. X. Cheng, “Coherent anti-Stokes Raman scattering imaging of axonal myelin in live spinal tissues,” Biophys. J.89(1), 581–591 (2005).
[CrossRef] [PubMed]

Grässel, D.

M. Axer, K. Amunts, D. Grässel, C. Palm, J. Dammers, H. Axer, U. Pietrzyk, and K. Zilles, “A novel approach to the human connectome: ultra-high resolution mapping of fiber tracts in the brain,” Neuroimage54(2), 1091–1101 (2011).
[CrossRef] [PubMed]

Hellwarth, R. W.

R. W. Hellwarth, “Third-order optical susceptibilities of liquids and solids,” Prog. Quantum Electron.5, 1–68 (1979).
[CrossRef]

Hsu, H.

C. Shang and H. Hsu, “The spatial symmetric forms of third-order nonlinear susceptibility,” IEEE J. Quantum Electron.23(2), 177–179 (1987).
[CrossRef]

Huff, T. B.

Kachynski, A. V.

A. V. Kachynski, A. N. Kuzmin, P. N. Prasad, and I. I. Smalyukh, “Coherent anti-Stokes Raman scattering polarized microscopy of three-dimensional director structures in liquid crystals,” Appl. Phys. Lett.91(15), 151905 (2007).
[CrossRef]

Kuzmin, A. N.

A. V. Kachynski, A. N. Kuzmin, P. N. Prasad, and I. I. Smalyukh, “Coherent anti-Stokes Raman scattering polarized microscopy of three-dimensional director structures in liquid crystals,” Appl. Phys. Lett.91(15), 151905 (2007).
[CrossRef]

Laffray, S.

Lin, C. P.

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.102(46), 16807–16812 (2005).
[CrossRef] [PubMed]

Maker, P.

P. Maker and R. Terhune, “Study of optical effects due to an induced polarization third order in the electric field strength,” Phys. Rev.137(3A), A801–A818 (1965).
[CrossRef]

Munhoz, F.

Palm, C.

M. Axer, K. Amunts, D. Grässel, C. Palm, J. Dammers, H. Axer, U. Pietrzyk, and K. Zilles, “A novel approach to the human connectome: ultra-high resolution mapping of fiber tracts in the brain,” Neuroimage54(2), 1091–1101 (2011).
[CrossRef] [PubMed]

Pautot, S.

J. X. Cheng, S. Pautot, D. A. Weitz, and X. S. Xie, “Ordering of water molecules between phospholipid bilayers visualized by coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.100(17), 9826–9830 (2003).
[CrossRef] [PubMed]

Pietrzyk, U.

M. Axer, K. Amunts, D. Grässel, C. Palm, J. Dammers, H. Axer, U. Pietrzyk, and K. Zilles, “A novel approach to the human connectome: ultra-high resolution mapping of fiber tracts in the brain,” Neuroimage54(2), 1091–1101 (2011).
[CrossRef] [PubMed]

Potma, E. O.

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.102(46), 16807–16812 (2005).
[CrossRef] [PubMed]

Prasad, P. N.

A. V. Kachynski, A. N. Kuzmin, P. N. Prasad, and I. I. Smalyukh, “Coherent anti-Stokes Raman scattering polarized microscopy of three-dimensional director structures in liquid crystals,” Appl. Phys. Lett.91(15), 151905 (2007).
[CrossRef]

Puoris’haag, M.

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.102(46), 16807–16812 (2005).
[CrossRef] [PubMed]

Rigneault, H.

Shang, C.

C. Shang and H. Hsu, “The spatial symmetric forms of third-order nonlinear susceptibility,” IEEE J. Quantum Electron.23(2), 177–179 (1987).
[CrossRef]

Shi, R.

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J. X. Cheng, “Coherent anti-Stokes Raman scattering imaging of axonal myelin in live spinal tissues,” Biophys. J.89(1), 581–591 (2005).
[CrossRef] [PubMed]

Smalyukh, I. I.

A. V. Kachynski, A. N. Kuzmin, P. N. Prasad, and I. I. Smalyukh, “Coherent anti-Stokes Raman scattering polarized microscopy of three-dimensional director structures in liquid crystals,” Appl. Phys. Lett.91(15), 151905 (2007).
[CrossRef]

Terhune, R.

P. Maker and R. Terhune, “Study of optical effects due to an induced polarization third order in the electric field strength,” Phys. Rev.137(3A), A801–A818 (1965).
[CrossRef]

Vallée, R.

Volkmer, A.

A. Volkmer, L. D. Book, and X. S. Xie, “Time-resolved coherent anti-Stokes Raman scattering microscopy: Imaging based on Raman free induction decay,” Appl. Phys. Lett.80(9), 1505–1507 (2002).
[CrossRef]

Wang, H.

Y. Fu, T. B. Huff, H.-W. Wang, J.-X. Cheng, and H. Wang, “Ex vivo and in vivo imaging of myelin fibers in mouse brain by coherent anti-Stokes Raman scattering microscopy,” Opt. Express16(24), 19396–19409 (2008).
[CrossRef] [PubMed]

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J. X. Cheng, “Coherent anti-Stokes Raman scattering imaging of axonal myelin in live spinal tissues,” Biophys. J.89(1), 581–591 (2005).
[CrossRef] [PubMed]

Wang, H.-W.

Weitz, D. A.

J. X. Cheng, S. Pautot, D. A. Weitz, and X. S. Xie, “Ordering of water molecules between phospholipid bilayers visualized by coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.100(17), 9826–9830 (2003).
[CrossRef] [PubMed]

Xie, X. S.

C. L. Evans and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu Rev Anal Chem (Palo Alto Calif)1(1), 883–909 (2008).
[CrossRef] [PubMed]

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.102(46), 16807–16812 (2005).
[CrossRef] [PubMed]

J. X. Cheng and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications,” J. Phys. Chem. B108(3), 827–840 (2004).
[CrossRef]

J. X. Cheng, S. Pautot, D. A. Weitz, and X. S. Xie, “Ordering of water molecules between phospholipid bilayers visualized by coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.100(17), 9826–9830 (2003).
[CrossRef] [PubMed]

A. Volkmer, L. D. Book, and X. S. Xie, “Time-resolved coherent anti-Stokes Raman scattering microscopy: Imaging based on Raman free induction decay,” Appl. Phys. Lett.80(9), 1505–1507 (2002).
[CrossRef]

J. X. Cheng, L. D. Book, and X. S. Xie, “Polarization coherent anti-Stokes Raman scattering microscopy,” Opt. Lett.26(17), 1341–1343 (2001).
[CrossRef] [PubMed]

Zickmund, P.

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J. X. Cheng, “Coherent anti-Stokes Raman scattering imaging of axonal myelin in live spinal tissues,” Biophys. J.89(1), 581–591 (2005).
[CrossRef] [PubMed]

Zilles, K.

M. Axer, K. Amunts, D. Grässel, C. Palm, J. Dammers, H. Axer, U. Pietrzyk, and K. Zilles, “A novel approach to the human connectome: ultra-high resolution mapping of fiber tracts in the brain,” Neuroimage54(2), 1091–1101 (2011).
[CrossRef] [PubMed]

Annu Rev Anal Chem (Palo Alto Calif)

C. L. Evans and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu Rev Anal Chem (Palo Alto Calif)1(1), 883–909 (2008).
[CrossRef] [PubMed]

Appl. Phys. Lett.

A. V. Kachynski, A. N. Kuzmin, P. N. Prasad, and I. I. Smalyukh, “Coherent anti-Stokes Raman scattering polarized microscopy of three-dimensional director structures in liquid crystals,” Appl. Phys. Lett.91(15), 151905 (2007).
[CrossRef]

A. Volkmer, L. D. Book, and X. S. Xie, “Time-resolved coherent anti-Stokes Raman scattering microscopy: Imaging based on Raman free induction decay,” Appl. Phys. Lett.80(9), 1505–1507 (2002).
[CrossRef]

Biophys. J.

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J. X. Cheng, “Coherent anti-Stokes Raman scattering imaging of axonal myelin in live spinal tissues,” Biophys. J.89(1), 581–591 (2005).
[CrossRef] [PubMed]

IEEE J. Quantum Electron.

C. Shang and H. Hsu, “The spatial symmetric forms of third-order nonlinear susceptibility,” IEEE J. Quantum Electron.23(2), 177–179 (1987).
[CrossRef]

J. Opt. Soc. Am. B

J. Phys. Chem. B

J. X. Cheng and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications,” J. Phys. Chem. B108(3), 827–840 (2004).
[CrossRef]

Neuroimage

M. Axer, K. Amunts, D. Grässel, C. Palm, J. Dammers, H. Axer, U. Pietrzyk, and K. Zilles, “A novel approach to the human connectome: ultra-high resolution mapping of fiber tracts in the brain,” Neuroimage54(2), 1091–1101 (2011).
[CrossRef] [PubMed]

M. D. Budde and J. A. Frank, “Examining brain microstructure using structure tensor analysis of histological sections,” Neuroimage63(1), 1–10 (2012).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Phys. Rev.

P. Maker and R. Terhune, “Study of optical effects due to an induced polarization third order in the electric field strength,” Phys. Rev.137(3A), A801–A818 (1965).
[CrossRef]

Proc. Natl. Acad. Sci. U.S.A.

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.102(46), 16807–16812 (2005).
[CrossRef] [PubMed]

J. X. Cheng, S. Pautot, D. A. Weitz, and X. S. Xie, “Ordering of water molecules between phospholipid bilayers visualized by coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A.100(17), 9826–9830 (2003).
[CrossRef] [PubMed]

Prog. Quantum Electron.

R. W. Hellwarth, “Third-order optical susceptibilities of liquids and solids,” Prog. Quantum Electron.5, 1–68 (1979).
[CrossRef]

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Figures (5)

Fig. 1
Fig. 1

Schematic representation of the RP-CARS setup. The 800-nm pulses from the laser (fs Laser), shown as red lines, are split by a beam splitter (BS) and routed to the supercontinuum generator (SCG) and, through two 3-nm bandpass filters (3-nm BP) centered at 800 nm, to the rotating λ/2 retarder (R-λ/2). The linearly-polarized broadband radiation (green lines) is delayed by a delay line (DL), transformed into circularly polarized light by a λ/4 retarder (λ/4), and recombined with the 800-nm radiation by means of a dichroic mirror (D). A second λ/4 retarder in the pump and probe path allows compensating for polarization distortions caused by D. The two are then routed to the high-numerical-aperture lens (Obj) through a pair of galvo-scanning mirrors, a scan lens and a tube lens. CARS signal is collected from the sample (S) by a condenser lens (Cond), band-pass filtered (BP), and routed to a photomultiplier tube (PMT). M0 to M2 are silver-coated mirrors. The output of the PMT is measured by phase-sensitive techniques by means of a lock-in amplifier. The reference phase and frequency for the lock-in amplifier is generated by a Hall sensor in close proximity to the rotor of the brushless motor that rotates R-λ/2.

Fig. 2
Fig. 2

Polarization-dependent artifact in a CARS image of a single nerve axon in transverse section (CH and CH2 bonds). (a) The polarization-dependent artifact is evident when linearly-polarized light is used. In this case the CH and CH2 chemical bonds aligned with the polarization plane of the incident light (vertical in the image) generate a stronger CARS signal. (b) Same as a) but using a perpendicular polarization plane of the incident light (horizontal in the image). The pump and probe beam and the Stokes beam power was respectively 30 mW and 12 mW. Scale bar: 0.5 μm.

Fig. 3
Fig. 3

RP-CARS imaging of the same single nerve axon as in Fig. 2 (CH2 bonds, transverse section). (a) Image constructed using A dc . Compared to the images in Fig. 2 it is free from the polarization artifacts. (b) Image constructed using A 2ω (bright pixels indicate that the signal value varies strongly as a function of the light polarization orientation). (c) Image of the phase of the signal collected from the same region (the colors indicate different light-polarization orientations). (d) RP-image constructed using the image in b) as brightness value and the one in c) as hue value. The myelin sheaths change color smoothly, covering all the possible orientations. The power levels were the same as in Fig. 2. The scale bar length is 0.5 μm.

Fig. 4
Fig. 4

Coronal section of the mouse brain anterior commissure imaged at different magnifications. This multi-scale acquisition shows the degree of local asymmetry in the direction of the acylic chains and their average spatial orientation (the color-coding scheme is described in the text). The white rectangles show the region magnified in the successive image of the series (indicated by an arrow). The colored lines in the top-left panel depict the color-to-orientation mapping. The last three images (*,§ and #) refer respectively to the three white rectangles in the preceding image. The pump and probe beam and the Stokes beam power was respectively 50 mW and 10 mW.

Fig. 5
Fig. 5

A dc and A 2ω signals of mouse brain slices (CH2 bonds). (a) Conventional CARS image (linar polarizations) from a 100-μm-thick brain slice (bregma: 0.4 mm). (b) A dc signal from a 150-μm-thick brain slice (bregma: −0.1 mm). Note that in (a) white matter (e.g., corpus callosum or anterior commissure) appears bright, whereas in (b) it appears dark. This effect is due to the different slice thickness: the thicker the slice, the more its dense white matter diffuses the CARS signal. (c) Image from the same brain slice shown in (b), but constructed using the A 2ω signal (divided by the A dc signal): in this case the white matter appears bright as in (a). The medial longitudinal fissure, filled with aqueous solution, appears artifactually bright due to its low A dc signal. cc: corpus callosum. ac: anterior commissure, its shape appears different in (a) with respect to (b) and (c) because of the diverse positioning of the coronal slice (distance to bregma). ml: medial longitudinal fissure. The pump and probe beam and the Stokes beam power was respectively 30 mW and 18 mW. Scale bar: 0.25 mm.

Equations (4)

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I CARS (t)= I p 2 I S [ A dc + A 2ω e (2iωt+iφ) + A 4ω e (4iωt+iφ) ],
A dc =3 c 11 2 +2 c 11 c 16 +14 c 16 2 +2 c 33 c 16 +3 c 33 2 A 2ω =4( c 11 2 c 33 2 ) A 4w = c 11 2 2 c 11 c 16 6 c 16 2 2 c 33 c 16 + c 33 2 .
χ ijkl δ ij δ kl + δ ik δ jl + δ il δ jk .
I CARS (t)= I p 2 I S [ B dc + B 2ω e (2iωt+iφ) ],

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