Abstract

The use of coherent anti-Stokes Raman scattering microscopy tuned to the lipid vibration for quantitative myelin imaging suffers from the excitation polarization dependence of this third-order nonlinear optical effect. The contrast obtained depends on the orientation of the myelin membrane, which in turn affects the morphometric parameters that can be extracted with image analysis. We show how circularly polarized laser beams can be used to avoid this complication, leading to images free of excitation polarization dependence. The technique promises to be optimal for in vivo imaging and the resulting images can be used for coherent anti-Stokes Raman scattering optical histology on native state tissue.

© 2009 Optical Society of America

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  5. B. N. G. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien, "The fluorescent toolbox for assessing protein location and function," Science 312, 217-224 (2006).
    [CrossRef] [PubMed]
  6. I. Veilleux, J. A. Spencer, D. P. Biss, D. Côté, and C. P. Lin, "In vivo cell tracking with video rate multimodality laser scanning microscopy," IEEE J. Sel. Top. Quantum Electron. 14, 10-18 (2008).
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  15. E. O. Potma and X. S. Xie, "Detection of single lipid bilayers with coherent anti-Stokes Raman scattering (CARS) microscopy," J. Raman Spectrosc. 34, 642-650 (2003).
    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  18. R. L. Friede and W. Beuche, "A new approach toward analyzing peripheral nerve fiber populations. I. Variance in sheath thickness corresponds to different geometric proportions of the internodes." J. Neuropathol. Exp. Neurol. 44, 60-72 (1985).
    [CrossRef] [PubMed]
  19. C. Hildebrand and R. Hahn, "Relation between myelin sheath thickness and axon size in spinal cord white matter of some vertebrate species," J. Neurol. Sci. 38, 421-434 (1978).
    [CrossRef] [PubMed]
  20. T. L. Mazely and W. M. H.III, "Third-order susceptibility tensors of partially ordered systems," J. Chem. Phys. 87, 1962-1966 (1987).
    [CrossRef]
  21. C. C. Shang and H. Hsu, "The spatial symmetrical forms of third-order nonlinear susceptibility," IEEE J. Quant. Electron. 23, 177-179 (1987).
    [CrossRef]
  22. D. A. Kleinman, "Nonlinear dielectric polarization in optical media," Phys. Rev. 126, 1977-1979 (1962).
    [CrossRef]
  23. D. Chemla, R. F. Begley, and R. Byer, "Experimental and theoretical studies of third-harmonic generation in the chalcopyrite CdGeAs2," IEEE J. Quantum Electron. 10, 71- 81 (1974).
    [CrossRef]
  24. S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, "Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914-3922 (2004).
    [CrossRef] [PubMed]
  25. C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, "Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation," J. Biomed. Opt. 13, 014005 (2008).
    [CrossRef] [PubMed]
  26. Y. Fu, T. B. Huff, H.-W. Wang, H. Wang, and J.-X. Cheng, "Ex vivo and in vivo imaging of myelin fibers in mouse brain by coherent anti-Stokes Raman scattering microscopy," Opt. Express 16, 19396-19409 (2008).
    [CrossRef] [PubMed]
  27. P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, "Polarization-modulated second harmonic generation in collagen," Biophys. J. 82, 3330-3342 (2002).
    [CrossRef] [PubMed]
  28. W. Beuche and R. L. Friede, "A new approach toward analyzing peripheral nerve fiber populations. II. Foreshortening of regenerated internodes corresponds to reduced sheath thickness." J. Neuropathol. Exp. Neurol. 44, 73-84 (1985).
    [CrossRef] [PubMed]
  29. R. S. Smith and Z. J. Koles, "Myelinated nerve fibers: computed effect of myelin thickness on conduction velocity," Am. J. Physiol. 219, 1256-1258 (1970).
    [PubMed]
  30. I. Micu, A. Ridsdale, L. Zhang, J. Woulfe, J. McClintock, C. A. Brantner, S. B. Andrews, and P. K. Stys, "Realtime measurement of free Ca2+ changes in CNS myelin by two-photon microscopy," Nat. Med. 13, 874-879 (2007).
    [CrossRef] [PubMed]

2009 (1)

F. P. Henry, D. Côté, M. A. Randolph, E. A. Z. Rust, R. W. Redmond, I. E. Kochevar, C. P. Lin, and J. M. Winograd, "Real-time in vivo assessment of the nerve microenvironment with coherent anti-Stokes Raman scattering microscopy," Plast. Reconstr. Surg. 123(2S), 123S-130S (2009).
[CrossRef]

2008 (4)

C. Evans and X. S. Xie, "Coherent Anti-Stokes Raman Scattering Microscopy: Chemical Imaging for Biology and Medicine," Annu. Rev. Anal. Chem. 1, 883-909 (2008).
[CrossRef]

I. Veilleux, J. A. Spencer, D. P. Biss, D. Côté, and C. P. Lin, "In vivo cell tracking with video rate multimodality laser scanning microscopy," IEEE J. Sel. Top. Quantum Electron. 14, 10-18 (2008).
[CrossRef]

C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, "Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation," J. Biomed. Opt. 13, 014005 (2008).
[CrossRef] [PubMed]

Y. Fu, T. B. Huff, H.-W. Wang, H. Wang, and J.-X. Cheng, "Ex vivo and in vivo imaging of myelin fibers in mouse brain by coherent anti-Stokes Raman scattering microscopy," Opt. Express 16, 19396-19409 (2008).
[CrossRef] [PubMed]

2007 (1)

I. Micu, A. Ridsdale, L. Zhang, J. Woulfe, J. McClintock, C. A. Brantner, S. B. Andrews, and P. K. Stys, "Realtime measurement of free Ca2+ changes in CNS myelin by two-photon microscopy," Nat. Med. 13, 874-879 (2007).
[CrossRef] [PubMed]

2006 (1)

B. N. G. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien, "The fluorescent toolbox for assessing protein location and function," Science 312, 217-224 (2006).
[CrossRef] [PubMed]

2005 (4)

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, "A guide to choosing fluorescent proteins," Nat. Methods 2, 905-909 (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, 16807-16812 (2005).
[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, 581-591 (2005).
[CrossRef] [PubMed]

2004 (3)

N. C. Shaner, R. E. Campbell, P. A. Steinbach, B. N. G. Giepmans, A. E. Palmer, and R. Y. Tsien, "Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein," Nat. Biotechnol. 22, 1567-1572 (2004).
[CrossRef] [PubMed]

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, "Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914-3922 (2004).
[CrossRef] [PubMed]

J.-X. Cheng and X. Xie, "Coherent Anti-Stokes Raman Scattering Microscopy: Instrumentation, Theory, and Applications," J. Phys. Chem. B 108, 827-840 (2004).
[CrossRef]

2003 (3)

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation." Proc. Natl. Acad. Sci. U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

P. J. Campagnola and L. M. Loew, "Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms," Nat. Biotechnol. 21, 1356-1360 (2003).
[CrossRef] [PubMed]

E. O. Potma and X. S. Xie, "Detection of single lipid bilayers with coherent anti-Stokes Raman scattering (CARS) microscopy," J. Raman Spectrosc. 34, 642-650 (2003).
[CrossRef]

2002 (1)

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, "Polarization-modulated second harmonic generation in collagen," Biophys. J. 82, 3330-3342 (2002).
[CrossRef] [PubMed]

2000 (1)

E. B. Hanlon, R. Manoharan, T. W. Koo, K. E. Shafer, J. T. Motz, M. Fitzmaurice, J. R. Kramer, I. Itzkan, R. R. Dasari, and M. S. Feld, "Prospects for in vivo Raman spectroscopy," Phys. Med. Biol. 45, R1-R59 (2000).
[CrossRef] [PubMed]

1987 (2)

T. L. Mazely and W. M. H.III, "Third-order susceptibility tensors of partially ordered systems," J. Chem. Phys. 87, 1962-1966 (1987).
[CrossRef]

C. C. Shang and H. Hsu, "The spatial symmetrical forms of third-order nonlinear susceptibility," IEEE J. Quant. Electron. 23, 177-179 (1987).
[CrossRef]

1986 (1)

R. L. Friede, "Computer editing of morphometric data on nerve fibers. An improved computer program," Acta Neuropathol. 72, 74-81 (1986).
[CrossRef] [PubMed]

1985 (3)

W. Beuche and R. L. Friede, "A quantitative assessment of myelin sheaths in the peripheral nerves of dystrophic, quaking, and trembler mutants." Acta Neuropathol. 66, 29-36 (1985).
[CrossRef] [PubMed]

R. L. Friede and W. Beuche, "A new approach toward analyzing peripheral nerve fiber populations. I. Variance in sheath thickness corresponds to different geometric proportions of the internodes." J. Neuropathol. Exp. Neurol. 44, 60-72 (1985).
[CrossRef] [PubMed]

W. Beuche and R. L. Friede, "A new approach toward analyzing peripheral nerve fiber populations. II. Foreshortening of regenerated internodes corresponds to reduced sheath thickness." J. Neuropathol. Exp. Neurol. 44, 73-84 (1985).
[CrossRef] [PubMed]

1978 (1)

C. Hildebrand and R. Hahn, "Relation between myelin sheath thickness and axon size in spinal cord white matter of some vertebrate species," J. Neurol. Sci. 38, 421-434 (1978).
[CrossRef] [PubMed]

1974 (1)

D. Chemla, R. F. Begley, and R. Byer, "Experimental and theoretical studies of third-harmonic generation in the chalcopyrite CdGeAs2," IEEE J. Quantum Electron. 10, 71- 81 (1974).
[CrossRef]

1970 (1)

R. S. Smith and Z. J. Koles, "Myelinated nerve fibers: computed effect of myelin thickness on conduction velocity," Am. J. Physiol. 219, 1256-1258 (1970).
[PubMed]

1962 (1)

D. A. Kleinman, "Nonlinear dielectric polarization in optical media," Phys. Rev. 126, 1977-1979 (1962).
[CrossRef]

Adams, S. R.

B. N. G. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien, "The fluorescent toolbox for assessing protein location and function," Science 312, 217-224 (2006).
[CrossRef] [PubMed]

Andrews, S. B.

I. Micu, A. Ridsdale, L. Zhang, J. Woulfe, J. McClintock, C. A. Brantner, S. B. Andrews, and P. K. Stys, "Realtime measurement of free Ca2+ changes in CNS myelin by two-photon microscopy," Nat. Med. 13, 874-879 (2007).
[CrossRef] [PubMed]

Begley, R. F.

D. Chemla, R. F. Begley, and R. Byer, "Experimental and theoretical studies of third-harmonic generation in the chalcopyrite CdGeAs2," IEEE J. Quantum Electron. 10, 71- 81 (1974).
[CrossRef]

Beuche, W.

W. Beuche and R. L. Friede, "A new approach toward analyzing peripheral nerve fiber populations. II. Foreshortening of regenerated internodes corresponds to reduced sheath thickness." J. Neuropathol. Exp. Neurol. 44, 73-84 (1985).
[CrossRef] [PubMed]

W. Beuche and R. L. Friede, "A quantitative assessment of myelin sheaths in the peripheral nerves of dystrophic, quaking, and trembler mutants." Acta Neuropathol. 66, 29-36 (1985).
[CrossRef] [PubMed]

R. L. Friede and W. Beuche, "A new approach toward analyzing peripheral nerve fiber populations. I. Variance in sheath thickness corresponds to different geometric proportions of the internodes." J. Neuropathol. Exp. Neurol. 44, 60-72 (1985).
[CrossRef] [PubMed]

Biss, D. P.

I. Veilleux, J. A. Spencer, D. P. Biss, D. Côté, and C. P. Lin, "In vivo cell tracking with video rate multimodality laser scanning microscopy," IEEE J. Sel. Top. Quantum Electron. 14, 10-18 (2008).
[CrossRef]

Brantner, C. A.

I. Micu, A. Ridsdale, L. Zhang, J. Woulfe, J. McClintock, C. A. Brantner, S. B. Andrews, and P. K. Stys, "Realtime measurement of free Ca2+ changes in CNS myelin by two-photon microscopy," Nat. Med. 13, 874-879 (2007).
[CrossRef] [PubMed]

Byer, R.

D. Chemla, R. F. Begley, and R. Byer, "Experimental and theoretical studies of third-harmonic generation in the chalcopyrite CdGeAs2," IEEE J. Quantum Electron. 10, 71- 81 (1974).
[CrossRef]

Campagnola, P. J.

P. J. Campagnola and L. M. Loew, "Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms," Nat. Biotechnol. 21, 1356-1360 (2003).
[CrossRef] [PubMed]

Campbell, R. E.

N. C. Shaner, R. E. Campbell, P. A. Steinbach, B. N. G. Giepmans, A. E. Palmer, and R. Y. Tsien, "Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein," Nat. Biotechnol. 22, 1567-1572 (2004).
[CrossRef] [PubMed]

Celliers, P. M.

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, "Polarization-modulated second harmonic generation in collagen," Biophys. J. 82, 3330-3342 (2002).
[CrossRef] [PubMed]

Chemla, D.

D. Chemla, R. F. Begley, and R. Byer, "Experimental and theoretical studies of third-harmonic generation in the chalcopyrite CdGeAs2," IEEE J. Quantum Electron. 10, 71- 81 (1974).
[CrossRef]

Chen, S.-Y.

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, "Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914-3922 (2004).
[CrossRef] [PubMed]

Chen, W.-L.

C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, "Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation," J. Biomed. Opt. 13, 014005 (2008).
[CrossRef] [PubMed]

Chen, Y.-C.

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, "Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914-3922 (2004).
[CrossRef] [PubMed]

Cheng, J.-X.

Y. Fu, T. B. Huff, H.-W. Wang, H. Wang, and J.-X. Cheng, "Ex vivo and in vivo imaging of myelin fibers in mouse brain by coherent anti-Stokes Raman scattering microscopy," Opt. Express 16, 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, 581-591 (2005).
[CrossRef] [PubMed]

J.-X. Cheng and X. Xie, "Coherent Anti-Stokes Raman Scattering Microscopy: Instrumentation, Theory, and Applications," J. Phys. Chem. B 108, 827-840 (2004).
[CrossRef]

Chern, G.-W.

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, "Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914-3922 (2004).
[CrossRef] [PubMed]

Chou, C.-K.

C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, "Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation," J. Biomed. Opt. 13, 014005 (2008).
[CrossRef] [PubMed]

Christie, R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation." Proc. Natl. Acad. Sci. U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Chu, S.-W.

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, "Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914-3922 (2004).
[CrossRef] [PubMed]

Côté, D.

F. P. Henry, D. Côté, M. A. Randolph, E. A. Z. Rust, R. W. Redmond, I. E. Kochevar, C. P. Lin, and J. M. Winograd, "Real-time in vivo assessment of the nerve microenvironment with coherent anti-Stokes Raman scattering microscopy," Plast. Reconstr. Surg. 123(2S), 123S-130S (2009).
[CrossRef]

I. Veilleux, J. A. Spencer, D. P. Biss, D. Côté, and C. P. Lin, "In vivo cell tracking with video rate multimodality laser scanning microscopy," IEEE J. Sel. Top. Quantum Electron. 14, 10-18 (2008).
[CrossRef]

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, 16807-16812 (2005).
[CrossRef] [PubMed]

Dasari, R. R.

E. B. Hanlon, R. Manoharan, T. W. Koo, K. E. Shafer, J. T. Motz, M. Fitzmaurice, J. R. Kramer, I. Itzkan, R. R. Dasari, and M. S. Feld, "Prospects for in vivo Raman spectroscopy," Phys. Med. Biol. 45, R1-R59 (2000).
[CrossRef] [PubMed]

Denk, W.

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

Dong, C.-Y.

C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, "Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation," J. Biomed. Opt. 13, 014005 (2008).
[CrossRef] [PubMed]

Ellisman, M. H.

B. N. G. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien, "The fluorescent toolbox for assessing protein location and function," Science 312, 217-224 (2006).
[CrossRef] [PubMed]

Evans, C.

C. Evans and X. S. Xie, "Coherent Anti-Stokes Raman Scattering Microscopy: Chemical Imaging for Biology and Medicine," Annu. Rev. Anal. Chem. 1, 883-909 (2008).
[CrossRef]

Evans, C. L.

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, 16807-16812 (2005).
[CrossRef] [PubMed]

Feld, M. S.

E. B. Hanlon, R. Manoharan, T. W. Koo, K. E. Shafer, J. T. Motz, M. Fitzmaurice, J. R. Kramer, I. Itzkan, R. R. Dasari, and M. S. Feld, "Prospects for in vivo Raman spectroscopy," Phys. Med. Biol. 45, R1-R59 (2000).
[CrossRef] [PubMed]

Fitzmaurice, M.

E. B. Hanlon, R. Manoharan, T. W. Koo, K. E. Shafer, J. T. Motz, M. Fitzmaurice, J. R. Kramer, I. Itzkan, R. R. Dasari, and M. S. Feld, "Prospects for in vivo Raman spectroscopy," Phys. Med. Biol. 45, R1-R59 (2000).
[CrossRef] [PubMed]

Friede, R. L.

R. L. Friede, "Computer editing of morphometric data on nerve fibers. An improved computer program," Acta Neuropathol. 72, 74-81 (1986).
[CrossRef] [PubMed]

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R. L. Friede and W. Beuche, "A new approach toward analyzing peripheral nerve fiber populations. I. Variance in sheath thickness corresponds to different geometric proportions of the internodes." J. Neuropathol. Exp. Neurol. 44, 60-72 (1985).
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W. Beuche and R. L. Friede, "A new approach toward analyzing peripheral nerve fiber populations. II. Foreshortening of regenerated internodes corresponds to reduced sheath thickness." J. Neuropathol. Exp. Neurol. 44, 73-84 (1985).
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Fu, Y.

Y. Fu, T. B. Huff, H.-W. Wang, H. Wang, and J.-X. Cheng, "Ex vivo and in vivo imaging of myelin fibers in mouse brain by coherent anti-Stokes Raman scattering microscopy," Opt. Express 16, 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, 581-591 (2005).
[CrossRef] [PubMed]

Fwu, P. T.

C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, "Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation," J. Biomed. Opt. 13, 014005 (2008).
[CrossRef] [PubMed]

Giepmans, B. N. G.

B. N. G. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien, "The fluorescent toolbox for assessing protein location and function," Science 312, 217-224 (2006).
[CrossRef] [PubMed]

N. C. Shaner, R. E. Campbell, P. A. Steinbach, B. N. G. Giepmans, A. E. Palmer, and R. Y. Tsien, "Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein," Nat. Biotechnol. 22, 1567-1572 (2004).
[CrossRef] [PubMed]

H., W. M.

T. L. Mazely and W. M. H.III, "Third-order susceptibility tensors of partially ordered systems," J. Chem. Phys. 87, 1962-1966 (1987).
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C. Hildebrand and R. Hahn, "Relation between myelin sheath thickness and axon size in spinal cord white matter of some vertebrate species," J. Neurol. Sci. 38, 421-434 (1978).
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E. B. Hanlon, R. Manoharan, T. W. Koo, K. E. Shafer, J. T. Motz, M. Fitzmaurice, J. R. Kramer, I. Itzkan, R. R. Dasari, and M. S. Feld, "Prospects for in vivo Raman spectroscopy," Phys. Med. Biol. 45, R1-R59 (2000).
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F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
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Henry, F. P.

F. P. Henry, D. Côté, M. A. Randolph, E. A. Z. Rust, R. W. Redmond, I. E. Kochevar, C. P. Lin, and J. M. Winograd, "Real-time in vivo assessment of the nerve microenvironment with coherent anti-Stokes Raman scattering microscopy," Plast. Reconstr. Surg. 123(2S), 123S-130S (2009).
[CrossRef]

Hildebrand, C.

C. Hildebrand and R. Hahn, "Relation between myelin sheath thickness and axon size in spinal cord white matter of some vertebrate species," J. Neurol. Sci. 38, 421-434 (1978).
[CrossRef] [PubMed]

Hsu, H.

C. C. Shang and H. Hsu, "The spatial symmetrical forms of third-order nonlinear susceptibility," IEEE J. Quant. Electron. 23, 177-179 (1987).
[CrossRef]

Huff, T. B.

Hyman, B. T.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation." Proc. Natl. Acad. Sci. U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Itzkan, I.

E. B. Hanlon, R. Manoharan, T. W. Koo, K. E. Shafer, J. T. Motz, M. Fitzmaurice, J. R. Kramer, I. Itzkan, R. R. Dasari, and M. S. Feld, "Prospects for in vivo Raman spectroscopy," Phys. Med. Biol. 45, R1-R59 (2000).
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D. A. Kleinman, "Nonlinear dielectric polarization in optical media," Phys. Rev. 126, 1977-1979 (1962).
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Kochevar, I. E.

F. P. Henry, D. Côté, M. A. Randolph, E. A. Z. Rust, R. W. Redmond, I. E. Kochevar, C. P. Lin, and J. M. Winograd, "Real-time in vivo assessment of the nerve microenvironment with coherent anti-Stokes Raman scattering microscopy," Plast. Reconstr. Surg. 123(2S), 123S-130S (2009).
[CrossRef]

Koles, Z. J.

R. S. Smith and Z. J. Koles, "Myelinated nerve fibers: computed effect of myelin thickness on conduction velocity," Am. J. Physiol. 219, 1256-1258 (1970).
[PubMed]

Koo, T. W.

E. B. Hanlon, R. Manoharan, T. W. Koo, K. E. Shafer, J. T. Motz, M. Fitzmaurice, J. R. Kramer, I. Itzkan, R. R. Dasari, and M. S. Feld, "Prospects for in vivo Raman spectroscopy," Phys. Med. Biol. 45, R1-R59 (2000).
[CrossRef] [PubMed]

Kramer, J. R.

E. B. Hanlon, R. Manoharan, T. W. Koo, K. E. Shafer, J. T. Motz, M. Fitzmaurice, J. R. Kramer, I. Itzkan, R. R. Dasari, and M. S. Feld, "Prospects for in vivo Raman spectroscopy," Phys. Med. Biol. 45, R1-R59 (2000).
[CrossRef] [PubMed]

Lee, H.-S.

C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, "Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation," J. Biomed. Opt. 13, 014005 (2008).
[CrossRef] [PubMed]

Lin, B.-L.

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, "Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914-3922 (2004).
[CrossRef] [PubMed]

Lin, C. P.

F. P. Henry, D. Côté, M. A. Randolph, E. A. Z. Rust, R. W. Redmond, I. E. Kochevar, C. P. Lin, and J. M. Winograd, "Real-time in vivo assessment of the nerve microenvironment with coherent anti-Stokes Raman scattering microscopy," Plast. Reconstr. Surg. 123(2S), 123S-130S (2009).
[CrossRef]

I. Veilleux, J. A. Spencer, D. P. Biss, D. Côté, and C. P. Lin, "In vivo cell tracking with video rate multimodality laser scanning microscopy," IEEE J. Sel. Top. Quantum Electron. 14, 10-18 (2008).
[CrossRef]

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, 16807-16812 (2005).
[CrossRef] [PubMed]

Lin, S.-J.

C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, "Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation," J. Biomed. Opt. 13, 014005 (2008).
[CrossRef] [PubMed]

Loew, L. M.

P. J. Campagnola and L. M. Loew, "Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms," Nat. Biotechnol. 21, 1356-1360 (2003).
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Manoharan, R.

E. B. Hanlon, R. Manoharan, T. W. Koo, K. E. Shafer, J. T. Motz, M. Fitzmaurice, J. R. Kramer, I. Itzkan, R. R. Dasari, and M. S. Feld, "Prospects for in vivo Raman spectroscopy," Phys. Med. Biol. 45, R1-R59 (2000).
[CrossRef] [PubMed]

Mazely, T. L.

T. L. Mazely and W. M. H.III, "Third-order susceptibility tensors of partially ordered systems," J. Chem. Phys. 87, 1962-1966 (1987).
[CrossRef]

McClintock, J.

I. Micu, A. Ridsdale, L. Zhang, J. Woulfe, J. McClintock, C. A. Brantner, S. B. Andrews, and P. K. Stys, "Realtime measurement of free Ca2+ changes in CNS myelin by two-photon microscopy," Nat. Med. 13, 874-879 (2007).
[CrossRef] [PubMed]

Micu, I.

I. Micu, A. Ridsdale, L. Zhang, J. Woulfe, J. McClintock, C. A. Brantner, S. B. Andrews, and P. K. Stys, "Realtime measurement of free Ca2+ changes in CNS myelin by two-photon microscopy," Nat. Med. 13, 874-879 (2007).
[CrossRef] [PubMed]

Motz, J. T.

E. B. Hanlon, R. Manoharan, T. W. Koo, K. E. Shafer, J. T. Motz, M. Fitzmaurice, J. R. Kramer, I. Itzkan, R. R. Dasari, and M. S. Feld, "Prospects for in vivo Raman spectroscopy," Phys. Med. Biol. 45, R1-R59 (2000).
[CrossRef] [PubMed]

Nikitin, A. Y.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation." Proc. Natl. Acad. Sci. U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Palmer, A. E.

N. C. Shaner, R. E. Campbell, P. A. Steinbach, B. N. G. Giepmans, A. E. Palmer, and R. Y. Tsien, "Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein," Nat. Biotechnol. 22, 1567-1572 (2004).
[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, 16807-16812 (2005).
[CrossRef] [PubMed]

E. O. Potma and X. S. Xie, "Detection of single lipid bilayers with coherent anti-Stokes Raman scattering (CARS) microscopy," J. Raman Spectrosc. 34, 642-650 (2003).
[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, 16807-16812 (2005).
[CrossRef] [PubMed]

Randolph, M. A.

F. P. Henry, D. Côté, M. A. Randolph, E. A. Z. Rust, R. W. Redmond, I. E. Kochevar, C. P. Lin, and J. M. Winograd, "Real-time in vivo assessment of the nerve microenvironment with coherent anti-Stokes Raman scattering microscopy," Plast. Reconstr. Surg. 123(2S), 123S-130S (2009).
[CrossRef]

Redmond, R. W.

F. P. Henry, D. Côté, M. A. Randolph, E. A. Z. Rust, R. W. Redmond, I. E. Kochevar, C. P. Lin, and J. M. Winograd, "Real-time in vivo assessment of the nerve microenvironment with coherent anti-Stokes Raman scattering microscopy," Plast. Reconstr. Surg. 123(2S), 123S-130S (2009).
[CrossRef]

Reiser, K. M.

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, "Polarization-modulated second harmonic generation in collagen," Biophys. J. 82, 3330-3342 (2002).
[CrossRef] [PubMed]

Ridsdale, A.

I. Micu, A. Ridsdale, L. Zhang, J. Woulfe, J. McClintock, C. A. Brantner, S. B. Andrews, and P. K. Stys, "Realtime measurement of free Ca2+ changes in CNS myelin by two-photon microscopy," Nat. Med. 13, 874-879 (2007).
[CrossRef] [PubMed]

Rubenchik, A. M.

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, "Polarization-modulated second harmonic generation in collagen," Biophys. J. 82, 3330-3342 (2002).
[CrossRef] [PubMed]

Rust, E. A. Z.

F. P. Henry, D. Côté, M. A. Randolph, E. A. Z. Rust, R. W. Redmond, I. E. Kochevar, C. P. Lin, and J. M. Winograd, "Real-time in vivo assessment of the nerve microenvironment with coherent anti-Stokes Raman scattering microscopy," Plast. Reconstr. Surg. 123(2S), 123S-130S (2009).
[CrossRef]

Shafer, K. E.

E. B. Hanlon, R. Manoharan, T. W. Koo, K. E. Shafer, J. T. Motz, M. Fitzmaurice, J. R. Kramer, I. Itzkan, R. R. Dasari, and M. S. Feld, "Prospects for in vivo Raman spectroscopy," Phys. Med. Biol. 45, R1-R59 (2000).
[CrossRef] [PubMed]

Shaner, N. C.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, "A guide to choosing fluorescent proteins," Nat. Methods 2, 905-909 (2005).
[CrossRef] [PubMed]

N. C. Shaner, R. E. Campbell, P. A. Steinbach, B. N. G. Giepmans, A. E. Palmer, and R. Y. Tsien, "Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein," Nat. Biotechnol. 22, 1567-1572 (2004).
[CrossRef] [PubMed]

Shang, C. C.

C. C. Shang and H. Hsu, "The spatial symmetrical forms of third-order nonlinear susceptibility," IEEE J. Quant. Electron. 23, 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, 581-591 (2005).
[CrossRef] [PubMed]

Smith, R. S.

R. S. Smith and Z. J. Koles, "Myelinated nerve fibers: computed effect of myelin thickness on conduction velocity," Am. J. Physiol. 219, 1256-1258 (1970).
[PubMed]

Spencer, J. A.

I. Veilleux, J. A. Spencer, D. P. Biss, D. Côté, and C. P. Lin, "In vivo cell tracking with video rate multimodality laser scanning microscopy," IEEE J. Sel. Top. Quantum Electron. 14, 10-18 (2008).
[CrossRef]

Steinbach, P. A.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, "A guide to choosing fluorescent proteins," Nat. Methods 2, 905-909 (2005).
[CrossRef] [PubMed]

N. C. Shaner, R. E. Campbell, P. A. Steinbach, B. N. G. Giepmans, A. E. Palmer, and R. Y. Tsien, "Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein," Nat. Biotechnol. 22, 1567-1572 (2004).
[CrossRef] [PubMed]

Stoller, P.

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, "Polarization-modulated second harmonic generation in collagen," Biophys. J. 82, 3330-3342 (2002).
[CrossRef] [PubMed]

Stys, P. K.

I. Micu, A. Ridsdale, L. Zhang, J. Woulfe, J. McClintock, C. A. Brantner, S. B. Andrews, and P. K. Stys, "Realtime measurement of free Ca2+ changes in CNS myelin by two-photon microscopy," Nat. Med. 13, 874-879 (2007).
[CrossRef] [PubMed]

Sun, C.-K.

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, "Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914-3922 (2004).
[CrossRef] [PubMed]

Tsai, T.-H.

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, "Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914-3922 (2004).
[CrossRef] [PubMed]

Tsien, R. Y.

B. N. G. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien, "The fluorescent toolbox for assessing protein location and function," Science 312, 217-224 (2006).
[CrossRef] [PubMed]

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, "A guide to choosing fluorescent proteins," Nat. Methods 2, 905-909 (2005).
[CrossRef] [PubMed]

N. C. Shaner, R. E. Campbell, P. A. Steinbach, B. N. G. Giepmans, A. E. Palmer, and R. Y. Tsien, "Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein," Nat. Biotechnol. 22, 1567-1572 (2004).
[CrossRef] [PubMed]

Veilleux, I.

I. Veilleux, J. A. Spencer, D. P. Biss, D. Côté, and C. P. Lin, "In vivo cell tracking with video rate multimodality laser scanning microscopy," IEEE J. Sel. Top. Quantum Electron. 14, 10-18 (2008).
[CrossRef]

Wang, H.

Y. Fu, T. B. Huff, H.-W. Wang, H. Wang, and J.-X. Cheng, "Ex vivo and in vivo imaging of myelin fibers in mouse brain by coherent anti-Stokes Raman scattering microscopy," Opt. Express 16, 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, 581-591 (2005).
[CrossRef] [PubMed]

Wang, H.-W.

Webb, W. W.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation." Proc. Natl. Acad. Sci. U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Williams, R. M.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation." Proc. Natl. Acad. Sci. U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Winograd, J. M.

F. P. Henry, D. Côté, M. A. Randolph, E. A. Z. Rust, R. W. Redmond, I. E. Kochevar, C. P. Lin, and J. M. Winograd, "Real-time in vivo assessment of the nerve microenvironment with coherent anti-Stokes Raman scattering microscopy," Plast. Reconstr. Surg. 123(2S), 123S-130S (2009).
[CrossRef]

Woulfe, J.

I. Micu, A. Ridsdale, L. Zhang, J. Woulfe, J. McClintock, C. A. Brantner, S. B. Andrews, and P. K. Stys, "Realtime measurement of free Ca2+ changes in CNS myelin by two-photon microscopy," Nat. Med. 13, 874-879 (2007).
[CrossRef] [PubMed]

Xie, X.

J.-X. Cheng and X. Xie, "Coherent Anti-Stokes Raman Scattering Microscopy: Instrumentation, Theory, and Applications," J. Phys. Chem. B 108, 827-840 (2004).
[CrossRef]

Xie, X. S.

C. Evans and X. S. Xie, "Coherent Anti-Stokes Raman Scattering Microscopy: Chemical Imaging for Biology and Medicine," Annu. Rev. Anal. Chem. 1, 883-909 (2008).
[CrossRef]

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, 16807-16812 (2005).
[CrossRef] [PubMed]

E. O. Potma and X. S. Xie, "Detection of single lipid bilayers with coherent anti-Stokes Raman scattering (CARS) microscopy," J. Raman Spectrosc. 34, 642-650 (2003).
[CrossRef]

Zhang, L.

I. Micu, A. Ridsdale, L. Zhang, J. Woulfe, J. McClintock, C. A. Brantner, S. B. Andrews, and P. K. Stys, "Realtime measurement of free Ca2+ changes in CNS myelin by two-photon microscopy," Nat. Med. 13, 874-879 (2007).
[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, 581-591 (2005).
[CrossRef] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation." Proc. Natl. Acad. Sci. U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Acta Neuropathol. (2)

R. L. Friede, "Computer editing of morphometric data on nerve fibers. An improved computer program," Acta Neuropathol. 72, 74-81 (1986).
[CrossRef] [PubMed]

W. Beuche and R. L. Friede, "A quantitative assessment of myelin sheaths in the peripheral nerves of dystrophic, quaking, and trembler mutants." Acta Neuropathol. 66, 29-36 (1985).
[CrossRef] [PubMed]

Am. J. Physiol. (1)

R. S. Smith and Z. J. Koles, "Myelinated nerve fibers: computed effect of myelin thickness on conduction velocity," Am. J. Physiol. 219, 1256-1258 (1970).
[PubMed]

Annu. Rev. Anal. Chem. (1)

C. Evans and X. S. Xie, "Coherent Anti-Stokes Raman Scattering Microscopy: Chemical Imaging for Biology and Medicine," Annu. Rev. Anal. Chem. 1, 883-909 (2008).
[CrossRef]

Biophys. J. (3)

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, 581-591 (2005).
[CrossRef] [PubMed]

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, "Polarization-modulated second harmonic generation in collagen," Biophys. J. 82, 3330-3342 (2002).
[CrossRef] [PubMed]

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, "Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914-3922 (2004).
[CrossRef] [PubMed]

IEEE J. Quant. Electron. (1)

C. C. Shang and H. Hsu, "The spatial symmetrical forms of third-order nonlinear susceptibility," IEEE J. Quant. Electron. 23, 177-179 (1987).
[CrossRef]

IEEE J. Quantum Electron. (1)

D. Chemla, R. F. Begley, and R. Byer, "Experimental and theoretical studies of third-harmonic generation in the chalcopyrite CdGeAs2," IEEE J. Quantum Electron. 10, 71- 81 (1974).
[CrossRef]

IEEE J. Sel. Top. Quantum Electron. (1)

I. Veilleux, J. A. Spencer, D. P. Biss, D. Côté, and C. P. Lin, "In vivo cell tracking with video rate multimodality laser scanning microscopy," IEEE J. Sel. Top. Quantum Electron. 14, 10-18 (2008).
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Supplementary Material (1)

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Figures (5)

Fig. 1.
Fig. 1.

(a) Laser system for CARS imaging. HWPp: half-wave plate at the Pump wavelength, HWPs: half-wave plate at the Stokes wavelength, QWPp: quarter-wave plate at the Pump wavelength, QWPs: quarter-wave plate at the Stokes wavelength, Tp: telescope for the Pump beam, Ts: telescope for the Stokes beam, DL: delay line, DF1: recombining dichroic filter, DF2: detection dichroic filter, MO: microscope objective, SPF: short-pass filter, BPF: band-pass filter, L: lens and PMT: photomultiplier tube. (b) Cross section view of a myelin sheath (only one wrapping is shown). Ep : Pump electric field amplitude, Es : Stokes electric field amplitude and θ is the angle between the laboratory x-axis and the direction of the linear polarization of the excitation beams. (c) Diagram of transverse and longitudinal imaging planes. (d) Evolution of the state of polarization of the excitation beams (either pump or Stokes) at the various positions labeled in (a). Dotted lines are the fast and slow axes of the HWP and QWP respectively. DF combines the effects of both dichroic filters DF1 and DF2.

Fig. 2.
Fig. 2.

Example of the excitation polarization dependence of the CARS signal of myelin. (a) and (b) Myelin images from transverse sections of fixed spinal cord, acquired with the excitation beams linearly polarized and circularly polarized respectively. (c) and (d) Myelin images from longitudinal sections of live spinal cord tissue at the equatorial plane, acquired with the excitation beams linearly polarized and circularly polarized respectively.

Fig. 3.
Fig. 3.

Quantification of the excitation polarization dependence of the CARS signal of a single cross section of myelinated axon from live spinal cord tissue. The images (a) and (b) have been acquired with the excitation beams linearly polarized along two perpendicular directions. (c) The expression for the theoretical excitation polarization dependence of the CARS signal intensity has been superimposed (black continuous line) to the experimentally measured polarization dependence (red dots).

Fig. 4.
Fig. 4.

A single cross section of myelin from a transverse slice of live (a) and fixed (c) spinal cord tissue are shown. (b) and (d) Illustrate the angular distribution of the normalized CARS signal for the myelin cross section shown in (a) and (c) respectively.

Fig. 5.
Fig. 5.

This set of curves displays structural informations from live spinal cord tissue with circularly polarized laser beams. (a) Typical contact-free CARS optical slice. (Media 1)(b) Histogram of caliber classes reveals that the mean axon diameter is 6.78±1.19 µm. (c) Scatter diagram of the g-ratio versus the axon caliber. The g-ratios varie between 0.5 and 0.65. (d) Plot of the axon diameter versus the fiber diameter. A linear regression reveals a mean g-ratio of 0.57±0.04. (e) Scatter diagram of myelin area to the fiber diameter. This single population curve shows that the probed nerve is in a non-pathological state.

Equations (4)

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

Iωaslinear (θ)Ep4Es2[sin2θ[c33+cos2θ[3c16c33]]2+
cos2 θ [3c16+cos2θ[c113c16]]2 ] ,
Iωascircular 18 Ep4 Es2 [[c11+3c16]2+[c333c16]2].
tan (2ϕ)=sin(2β)tan(δ),

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