Abstract

The second-harmonic signal in collagen, even in highly organized samples such as rat tail tendon fascicles, varies significantly with position. Previous studies suggest that this variability may be due to the parallel and antiparallel orientation of neighboring collagen fibrils. We applied high-resolution second-harmonic generation microscopy to confirm this hypothesis. Studies in which the focal spot diameter was varied from ∼1 to ∼6 μm strongly suggest that regions in which collagen fibrils have the same orientation in rat tail tendon are likely to be less than ∼1 μm in diameter. These measurements required accurate determination of the focal spot size achieved by use of different microscope objectives; we developed a technique that uses second-harmonic generation in a quartz reference to measure the focal spot diameter directly. We also used the quartz reference to determine a lower limit (d XXX > 0.4 pm/V) for the magnitude of the second-order nonlinear susceptibility in collagen.

© 2003 Optical Society of America

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  1. P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6, 277–286 (2001).
    [CrossRef] [PubMed]
  2. P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 81, 493–508 (2002).
    [CrossRef]
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    [CrossRef]
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    [CrossRef] [PubMed]
  11. P. Stoller, K. M. Reiser, P. M. Celliers, A. M. Rubenchik, “Polarization-modulated second harmonic generation in collagen,” Biophys. J. 82, 3330–3342 (2002).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
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  24. D. J. Maitland, “Dynamic measurements of tissue birefringence: theory and experiments,” Ph.D. dissertation (Northwestern University, Evanston, Ill., 1995).
  25. R. G. Byer, “Parametric oscillators and nonlinear materials,” in Nonlinear Optics, P. G. Harper, B. S. Wherrett, eds. (Academic, London, 1977), pp. 47–160.
  26. R. W. Boyd, Nonlinear Optics (Academic, San Diego, Calif., 1992).
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    [CrossRef] [PubMed]
  28. Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
    [CrossRef]
  29. J. M. Schins, G. J. Brakenhoff, M. Müller, “Characterizing layered structures with third-harmonic generation microscopy,” GIT Imag. Microsc. 1, 44–46 (2002).
  30. G. Ghosh, “Dispersion-equation coefficients for the refractive index and birefringence of calcite and quartz crystals,” Opt. Commun. 163, 95–102 (1999).
    [CrossRef]
  31. R. Gauderon, P. B. Lukins, C. J. R. Sheppard, “Optimization of second-harmonic generation microscopy,” Micron 32, 691–700 (2001).
    [CrossRef] [PubMed]
  32. E. Baer, J. J. Cassidy, A. Hiltner, “Hierarchical structure of collagen and its relationship to the physical properties of tendon,” in Collagen: Biochemistry and Biomechanics, M. E. Nimni, ed. (CRC, Boca Raton, Fla., 1988), Vol. 2, pp. 177–199.

2002 (5)

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 81, 493–508 (2002).
[CrossRef]

P. Stoller, B.-M. Kim, K. M. Reiser, A. M. Rubenchik, L. B. Da Silva, “Polarization dependent optical second harmonic imaging of rat-tail tendon,” J. Biomed. Opt. 7, 205–214 (2002).
[CrossRef] [PubMed]

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

A. Zoumi, A. Yeh, B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. USA 99, 11014–11019 (2002).
[CrossRef] [PubMed]

J. M. Schins, G. J. Brakenhoff, M. Müller, “Characterizing layered structures with third-harmonic generation microscopy,” GIT Imag. Microsc. 1, 44–46 (2002).

2001 (6)

R. Gauderon, P. B. Lukins, C. J. R. Sheppard, “Optimization of second-harmonic generation microscopy,” Micron 32, 691–700 (2001).
[CrossRef] [PubMed]

L. Moreaux, O. Sandre, S. Charpak, M. Blanchard-Desce, J. Mertz, “Coherent scattering in multi-harmonic light microscopy,” Biophys. J. 80, 1568–1574 (2001).
[CrossRef] [PubMed]

R. M. Williams, W. R. Zipfel, W. W. Webb, “Multiphoton microscopy in biological research,” Curr. Opin. Chem. Biol. 5, 603–608 (2001).
[CrossRef] [PubMed]

P.-C. Cheng, C.-K. Sun, B. L. Lin, F.-J. Kao, S.-W. Chu, “Biological multi-modality nonlinear spectromicroscopy: multiphoton fluorescence, second- and third-harmonic generation,” Scanning 23, 109–110 (2001), http://www.scanning.org .

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6, 277–286 (2001).
[CrossRef] [PubMed]

J. Squier, M. Müller, “High resolution nonlinear microscopy: a review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum. 72, 2855–2867 (2001).
[CrossRef]

2000 (3)

B.-M. Kim, J. Eichler, K. M. Reiser, A. M. Rubenchik, L. B. Da Silva, “Collagen structure and nonlinear susceptibility: effect of heat, glycation, and enzymatic cleavage on second harmonic signal intensity,” Lasers Surg. Med. 27, 329–335 (2000).
[CrossRef]

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200, 83–104 (2000).
[CrossRef] [PubMed]

L. Moreaux, O. Sandre, J. Mertz, “Membrane imaging by second-harmonic generation microscopy,” J. Opt. Soc. Am. B 17, 1685–1694 (2000).
[CrossRef]

1999 (3)

G. Ghosh, “Dispersion-equation coefficients for the refractive index and birefringence of calcite and quartz crystals,” Opt. Commun. 163, 95–102 (1999).
[CrossRef]

B.-M. Kim, J. Eichler, L. B. Da Silva, “Frequency doubling of ultrashort laser pulses in biological tissues,” Appl. Opt. 38, 7145–7150 (1999).
[CrossRef]

P. J. Campagnola, M. Wei, A. Lewis, L. M. Loew, “High-resolution nonlinear optical imaging of live cells by second harmonic generation,” Biophys. J. 77, 3341–3349 (1999).
[CrossRef] [PubMed]

1997 (2)

Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu, N. Zhadin, R. R. Alfano, “Second-harmonic tomography of tissue,” Opt. Lett. 22, 1323–1325 (1997).
[CrossRef]

Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

1996 (1)

1989 (1)

1986 (2)

I. Freund, M. Deutsch, “Macroscopic polarity of connective tissue is due to discrete polar structures,” Biopolymers 25, 601–606 (1986).
[CrossRef] [PubMed]

I. Freund, M. Deutsch, A. Sprecher, “Connective tissue polarity: optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon,” Biophys. J. 50, 693–712 (1986).
[CrossRef] [PubMed]

1981 (1)

S. Roth, I. Freund, “Optical second-harmonic scattering in rat-tail tendon,” Biopolymers 20, 1271–1290 (1981).
[CrossRef] [PubMed]

1977 (1)

D. A. D. Parry, A. S. Craig, “Quantitative electron microscope observations of the collagen fibrils in rat-tail tendon,” Biopolymers 16, 1015–1031 (1977).
[CrossRef] [PubMed]

1971 (1)

Alfano, R. R.

Baer, E.

E. Baer, J. J. Cassidy, A. Hiltner, “Hierarchical structure of collagen and its relationship to the physical properties of tendon,” in Collagen: Biochemistry and Biomechanics, M. E. Nimni, ed. (CRC, Boca Raton, Fla., 1988), Vol. 2, pp. 177–199.

Barad, Y.

Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

Blanchard-Desce, M.

L. Moreaux, O. Sandre, S. Charpak, M. Blanchard-Desce, J. Mertz, “Coherent scattering in multi-harmonic light microscopy,” Biophys. J. 80, 1568–1574 (2001).
[CrossRef] [PubMed]

Bolin, F. P.

Boyd, R. W.

R. W. Boyd, Nonlinear Optics (Academic, San Diego, Calif., 1992).

Brakenhoff, G. J.

J. M. Schins, G. J. Brakenhoff, M. Müller, “Characterizing layered structures with third-harmonic generation microscopy,” GIT Imag. Microsc. 1, 44–46 (2002).

Byer, R. G.

R. G. Byer, “Parametric oscillators and nonlinear materials,” in Nonlinear Optics, P. G. Harper, B. S. Wherrett, eds. (Academic, London, 1977), pp. 47–160.

Campagnola, P. J.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 81, 493–508 (2002).
[CrossRef]

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6, 277–286 (2001).
[CrossRef] [PubMed]

P. J. Campagnola, M. Wei, A. Lewis, L. M. Loew, “High-resolution nonlinear optical imaging of live cells by second harmonic generation,” Biophys. J. 77, 3341–3349 (1999).
[CrossRef] [PubMed]

Cassidy, J. J.

E. Baer, J. J. Cassidy, A. Hiltner, “Hierarchical structure of collagen and its relationship to the physical properties of tendon,” in Collagen: Biochemistry and Biomechanics, M. E. Nimni, ed. (CRC, Boca Raton, Fla., 1988), Vol. 2, pp. 177–199.

Celliers, P. M.

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

Charpak, S.

L. Moreaux, O. Sandre, S. Charpak, M. Blanchard-Desce, J. Mertz, “Coherent scattering in multi-harmonic light microscopy,” Biophys. J. 80, 1568–1574 (2001).
[CrossRef] [PubMed]

Cheng, P.-C.

P.-C. Cheng, C.-K. Sun, B. L. Lin, F.-J. Kao, S.-W. Chu, “Biological multi-modality nonlinear spectromicroscopy: multiphoton fluorescence, second- and third-harmonic generation,” Scanning 23, 109–110 (2001), http://www.scanning.org .

Chu, S.-W.

P.-C. Cheng, C.-K. Sun, B. L. Lin, F.-J. Kao, S.-W. Chu, “Biological multi-modality nonlinear spectromicroscopy: multiphoton fluorescence, second- and third-harmonic generation,” Scanning 23, 109–110 (2001), http://www.scanning.org .

Clark, H. A.

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6, 277–286 (2001).
[CrossRef] [PubMed]

Craig, A. S.

D. A. D. Parry, A. S. Craig, “Quantitative electron microscope observations of the collagen fibrils in rat-tail tendon,” Biopolymers 16, 1015–1031 (1977).
[CrossRef] [PubMed]

Da Silva, L. B.

P. Stoller, B.-M. Kim, K. M. Reiser, A. M. Rubenchik, L. B. Da Silva, “Polarization dependent optical second harmonic imaging of rat-tail tendon,” J. Biomed. Opt. 7, 205–214 (2002).
[CrossRef] [PubMed]

B.-M. Kim, J. Eichler, K. M. Reiser, A. M. Rubenchik, L. B. Da Silva, “Collagen structure and nonlinear susceptibility: effect of heat, glycation, and enzymatic cleavage on second harmonic signal intensity,” Lasers Surg. Med. 27, 329–335 (2000).
[CrossRef]

B.-M. Kim, J. Eichler, L. B. Da Silva, “Frequency doubling of ultrashort laser pulses in biological tissues,” Appl. Opt. 38, 7145–7150 (1999).
[CrossRef]

Deutsch, M.

I. Freund, M. Deutsch, “Macroscopic polarity of connective tissue is due to discrete polar structures,” Biopolymers 25, 601–606 (1986).
[CrossRef] [PubMed]

I. Freund, M. Deutsch, A. Sprecher, “Connective tissue polarity: optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon,” Biophys. J. 50, 693–712 (1986).
[CrossRef] [PubMed]

Eichler, J.

B.-M. Kim, J. Eichler, K. M. Reiser, A. M. Rubenchik, L. B. Da Silva, “Collagen structure and nonlinear susceptibility: effect of heat, glycation, and enzymatic cleavage on second harmonic signal intensity,” Lasers Surg. Med. 27, 329–335 (2000).
[CrossRef]

B.-M. Kim, J. Eichler, L. B. Da Silva, “Frequency doubling of ultrashort laser pulses in biological tissues,” Appl. Opt. 38, 7145–7150 (1999).
[CrossRef]

Eisenberg, H.

Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

Ference, R. J.

Fine, S.

Freund, I.

I. Freund, M. Deutsch, A. Sprecher, “Connective tissue polarity: optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon,” Biophys. J. 50, 693–712 (1986).
[CrossRef] [PubMed]

I. Freund, M. Deutsch, “Macroscopic polarity of connective tissue is due to discrete polar structures,” Biopolymers 25, 601–606 (1986).
[CrossRef] [PubMed]

S. Roth, I. Freund, “Optical second-harmonic scattering in rat-tail tendon,” Biopolymers 20, 1271–1290 (1981).
[CrossRef] [PubMed]

Gauderon, R.

R. Gauderon, P. B. Lukins, C. J. R. Sheppard, “Optimization of second-harmonic generation microscopy,” Micron 32, 691–700 (2001).
[CrossRef] [PubMed]

Ghosh, G.

G. Ghosh, “Dispersion-equation coefficients for the refractive index and birefringence of calcite and quartz crystals,” Opt. Commun. 163, 95–102 (1999).
[CrossRef]

Guo, Y.

Hansen, W. P.

Harris, D.

Hiltner, A.

E. Baer, J. J. Cassidy, A. Hiltner, “Hierarchical structure of collagen and its relationship to the physical properties of tendon,” in Collagen: Biochemistry and Biomechanics, M. E. Nimni, ed. (CRC, Boca Raton, Fla., 1988), Vol. 2, pp. 177–199.

Ho, P. P.

Hoppe, P. E.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 81, 493–508 (2002).
[CrossRef]

Horowitz, M.

Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

Hovanessian, V.

V. Hovanessian, A. Lalayan, “Second harmonic generation in biofiber-containing tissue,” Proceedings of the International Conference on Lasers 1996, V. J. Corcoran, T. A. Goldman, eds. (STS, McClean, Va., 1997), pp. 107–109.

Kao, F.-J.

P.-C. Cheng, C.-K. Sun, B. L. Lin, F.-J. Kao, S.-W. Chu, “Biological multi-modality nonlinear spectromicroscopy: multiphoton fluorescence, second- and third-harmonic generation,” Scanning 23, 109–110 (2001), http://www.scanning.org .

Kim, B.-M.

P. Stoller, B.-M. Kim, K. M. Reiser, A. M. Rubenchik, L. B. Da Silva, “Polarization dependent optical second harmonic imaging of rat-tail tendon,” J. Biomed. Opt. 7, 205–214 (2002).
[CrossRef] [PubMed]

B.-M. Kim, J. Eichler, K. M. Reiser, A. M. Rubenchik, L. B. Da Silva, “Collagen structure and nonlinear susceptibility: effect of heat, glycation, and enzymatic cleavage on second harmonic signal intensity,” Lasers Surg. Med. 27, 329–335 (2000).
[CrossRef]

B.-M. Kim, J. Eichler, L. B. Da Silva, “Frequency doubling of ultrashort laser pulses in biological tissues,” Appl. Opt. 38, 7145–7150 (1999).
[CrossRef]

König, K.

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200, 83–104 (2000).
[CrossRef] [PubMed]

Lalayan, A.

V. Hovanessian, A. Lalayan, “Second harmonic generation in biofiber-containing tissue,” Proceedings of the International Conference on Lasers 1996, V. J. Corcoran, T. A. Goldman, eds. (STS, McClean, Va., 1997), pp. 107–109.

Lewis, A.

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6, 277–286 (2001).
[CrossRef] [PubMed]

P. J. Campagnola, M. Wei, A. Lewis, L. M. Loew, “High-resolution nonlinear optical imaging of live cells by second harmonic generation,” Biophys. J. 77, 3341–3349 (1999).
[CrossRef] [PubMed]

Lin, B. L.

P.-C. Cheng, C.-K. Sun, B. L. Lin, F.-J. Kao, S.-W. Chu, “Biological multi-modality nonlinear spectromicroscopy: multiphoton fluorescence, second- and third-harmonic generation,” Scanning 23, 109–110 (2001), http://www.scanning.org .

Liu, F.

Loew, L. M.

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6, 277–286 (2001).
[CrossRef] [PubMed]

P. J. Campagnola, M. Wei, A. Lewis, L. M. Loew, “High-resolution nonlinear optical imaging of live cells by second harmonic generation,” Biophys. J. 77, 3341–3349 (1999).
[CrossRef] [PubMed]

Lui, F.

Lukins, P. B.

R. Gauderon, P. B. Lukins, C. J. R. Sheppard, “Optimization of second-harmonic generation microscopy,” Micron 32, 691–700 (2001).
[CrossRef] [PubMed]

Maitland, D. J.

D. J. Maitland, “Dynamic measurements of tissue birefringence: theory and experiments,” Ph.D. dissertation (Northwestern University, Evanston, Ill., 1995).

Malone, C. J.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 81, 493–508 (2002).
[CrossRef]

Mertz, J.

L. Moreaux, O. Sandre, S. Charpak, M. Blanchard-Desce, J. Mertz, “Coherent scattering in multi-harmonic light microscopy,” Biophys. J. 80, 1568–1574 (2001).
[CrossRef] [PubMed]

L. Moreaux, O. Sandre, J. Mertz, “Membrane imaging by second-harmonic generation microscopy,” J. Opt. Soc. Am. B 17, 1685–1694 (2000).
[CrossRef]

Millard, A. C.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 81, 493–508 (2002).
[CrossRef]

Mohler, W. A.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 81, 493–508 (2002).
[CrossRef]

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6, 277–286 (2001).
[CrossRef] [PubMed]

Moreaux, L.

L. Moreaux, O. Sandre, S. Charpak, M. Blanchard-Desce, J. Mertz, “Coherent scattering in multi-harmonic light microscopy,” Biophys. J. 80, 1568–1574 (2001).
[CrossRef] [PubMed]

L. Moreaux, O. Sandre, J. Mertz, “Membrane imaging by second-harmonic generation microscopy,” J. Opt. Soc. Am. B 17, 1685–1694 (2000).
[CrossRef]

Müller, M.

J. M. Schins, G. J. Brakenhoff, M. Müller, “Characterizing layered structures with third-harmonic generation microscopy,” GIT Imag. Microsc. 1, 44–46 (2002).

J. Squier, M. Müller, “High resolution nonlinear microscopy: a review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum. 72, 2855–2867 (2001).
[CrossRef]

Parry, D. A. D.

D. A. D. Parry, A. S. Craig, “Quantitative electron microscope observations of the collagen fibrils in rat-tail tendon,” Biopolymers 16, 1015–1031 (1977).
[CrossRef] [PubMed]

Poh, D. T.

D. T. Poh, “Examination of refractive index of human epidermis in-vitro and in-vivo,” in Proceedings of the International Conference on Lasers ’96, V. J. Corcoran, T. A. Goldman, eds. (STS, McLean, Va., 1997), pp. 118–125.

Preuss, L. E.

Reiser, K. M.

P. Stoller, B.-M. Kim, K. M. Reiser, A. M. Rubenchik, L. B. Da Silva, “Polarization dependent optical second harmonic imaging of rat-tail tendon,” J. Biomed. Opt. 7, 205–214 (2002).
[CrossRef] [PubMed]

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

B.-M. Kim, J. Eichler, K. M. Reiser, A. M. Rubenchik, L. B. Da Silva, “Collagen structure and nonlinear susceptibility: effect of heat, glycation, and enzymatic cleavage on second harmonic signal intensity,” Lasers Surg. Med. 27, 329–335 (2000).
[CrossRef]

Roth, S.

S. Roth, I. Freund, “Optical second-harmonic scattering in rat-tail tendon,” Biopolymers 20, 1271–1290 (1981).
[CrossRef] [PubMed]

Rubenchik, A. M.

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

P. Stoller, B.-M. Kim, K. M. Reiser, A. M. Rubenchik, L. B. Da Silva, “Polarization dependent optical second harmonic imaging of rat-tail tendon,” J. Biomed. Opt. 7, 205–214 (2002).
[CrossRef] [PubMed]

B.-M. Kim, J. Eichler, K. M. Reiser, A. M. Rubenchik, L. B. Da Silva, “Collagen structure and nonlinear susceptibility: effect of heat, glycation, and enzymatic cleavage on second harmonic signal intensity,” Lasers Surg. Med. 27, 329–335 (2000).
[CrossRef]

Sacks, P.

Sandre, O.

L. Moreaux, O. Sandre, S. Charpak, M. Blanchard-Desce, J. Mertz, “Coherent scattering in multi-harmonic light microscopy,” Biophys. J. 80, 1568–1574 (2001).
[CrossRef] [PubMed]

L. Moreaux, O. Sandre, J. Mertz, “Membrane imaging by second-harmonic generation microscopy,” J. Opt. Soc. Am. B 17, 1685–1694 (2000).
[CrossRef]

Savage, H.

Schantz, S.

Schins, J. M.

J. M. Schins, G. J. Brakenhoff, M. Müller, “Characterizing layered structures with third-harmonic generation microscopy,” GIT Imag. Microsc. 1, 44–46 (2002).

Sheppard, C. J. R.

R. Gauderon, P. B. Lukins, C. J. R. Sheppard, “Optimization of second-harmonic generation microscopy,” Micron 32, 691–700 (2001).
[CrossRef] [PubMed]

Silberberg, Y.

Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

Sprecher, A.

I. Freund, M. Deutsch, A. Sprecher, “Connective tissue polarity: optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon,” Biophys. J. 50, 693–712 (1986).
[CrossRef] [PubMed]

Squier, J.

J. Squier, M. Müller, “High resolution nonlinear microscopy: a review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum. 72, 2855–2867 (2001).
[CrossRef]

Stoller, P.

P. Stoller, B.-M. Kim, K. M. Reiser, A. M. Rubenchik, L. B. Da Silva, “Polarization dependent optical second harmonic imaging of rat-tail tendon,” J. Biomed. Opt. 7, 205–214 (2002).
[CrossRef] [PubMed]

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

Sun, C.-K.

P.-C. Cheng, C.-K. Sun, B. L. Lin, F.-J. Kao, S.-W. Chu, “Biological multi-modality nonlinear spectromicroscopy: multiphoton fluorescence, second- and third-harmonic generation,” Scanning 23, 109–110 (2001), http://www.scanning.org .

Taylor, R. C.

Terasaki, M.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 81, 493–508 (2002).
[CrossRef]

Tirksliunas, A.

Tromberg, B. J.

A. Zoumi, A. Yeh, B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. USA 99, 11014–11019 (2002).
[CrossRef] [PubMed]

Webb, W. W.

R. M. Williams, W. R. Zipfel, W. W. Webb, “Multiphoton microscopy in biological research,” Curr. Opin. Chem. Biol. 5, 603–608 (2001).
[CrossRef] [PubMed]

Wei, M.

P. J. Campagnola, M. Wei, A. Lewis, L. M. Loew, “High-resolution nonlinear optical imaging of live cells by second harmonic generation,” Biophys. J. 77, 3341–3349 (1999).
[CrossRef] [PubMed]

Williams, R. M.

R. M. Williams, W. R. Zipfel, W. W. Webb, “Multiphoton microscopy in biological research,” Curr. Opin. Chem. Biol. 5, 603–608 (2001).
[CrossRef] [PubMed]

Yeh, A.

A. Zoumi, A. Yeh, B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. USA 99, 11014–11019 (2002).
[CrossRef] [PubMed]

Zhadin, N.

Zipfel, W. R.

R. M. Williams, W. R. Zipfel, W. W. Webb, “Multiphoton microscopy in biological research,” Curr. Opin. Chem. Biol. 5, 603–608 (2001).
[CrossRef] [PubMed]

Zoumi, A.

A. Zoumi, A. Yeh, B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. USA 99, 11014–11019 (2002).
[CrossRef] [PubMed]

Appl. Opt. (4)

Appl. Phys. Lett. (1)

Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

Biophys. J. (5)

I. Freund, M. Deutsch, A. Sprecher, “Connective tissue polarity: optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon,” Biophys. J. 50, 693–712 (1986).
[CrossRef] [PubMed]

P. J. Campagnola, M. Wei, A. Lewis, L. M. Loew, “High-resolution nonlinear optical imaging of live cells by second harmonic generation,” Biophys. J. 77, 3341–3349 (1999).
[CrossRef] [PubMed]

L. Moreaux, O. Sandre, S. Charpak, M. Blanchard-Desce, J. Mertz, “Coherent scattering in multi-harmonic light microscopy,” Biophys. J. 80, 1568–1574 (2001).
[CrossRef] [PubMed]

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

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 81, 493–508 (2002).
[CrossRef]

Biopolymers (3)

S. Roth, I. Freund, “Optical second-harmonic scattering in rat-tail tendon,” Biopolymers 20, 1271–1290 (1981).
[CrossRef] [PubMed]

I. Freund, M. Deutsch, “Macroscopic polarity of connective tissue is due to discrete polar structures,” Biopolymers 25, 601–606 (1986).
[CrossRef] [PubMed]

D. A. D. Parry, A. S. Craig, “Quantitative electron microscope observations of the collagen fibrils in rat-tail tendon,” Biopolymers 16, 1015–1031 (1977).
[CrossRef] [PubMed]

Curr. Opin. Chem. Biol. (1)

R. M. Williams, W. R. Zipfel, W. W. Webb, “Multiphoton microscopy in biological research,” Curr. Opin. Chem. Biol. 5, 603–608 (2001).
[CrossRef] [PubMed]

GIT Imag. Microsc. (1)

J. M. Schins, G. J. Brakenhoff, M. Müller, “Characterizing layered structures with third-harmonic generation microscopy,” GIT Imag. Microsc. 1, 44–46 (2002).

J. Biomed. Opt. (2)

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6, 277–286 (2001).
[CrossRef] [PubMed]

P. Stoller, B.-M. Kim, K. M. Reiser, A. M. Rubenchik, L. B. Da Silva, “Polarization dependent optical second harmonic imaging of rat-tail tendon,” J. Biomed. Opt. 7, 205–214 (2002).
[CrossRef] [PubMed]

J. Microsc. (1)

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200, 83–104 (2000).
[CrossRef] [PubMed]

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

Lasers Surg. Med. (1)

B.-M. Kim, J. Eichler, K. M. Reiser, A. M. Rubenchik, L. B. Da Silva, “Collagen structure and nonlinear susceptibility: effect of heat, glycation, and enzymatic cleavage on second harmonic signal intensity,” Lasers Surg. Med. 27, 329–335 (2000).
[CrossRef]

Micron (1)

R. Gauderon, P. B. Lukins, C. J. R. Sheppard, “Optimization of second-harmonic generation microscopy,” Micron 32, 691–700 (2001).
[CrossRef] [PubMed]

Opt. Commun. (1)

G. Ghosh, “Dispersion-equation coefficients for the refractive index and birefringence of calcite and quartz crystals,” Opt. Commun. 163, 95–102 (1999).
[CrossRef]

Opt. Lett. (1)

Proc. Natl. Acad. Sci. USA (1)

A. Zoumi, A. Yeh, B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. USA 99, 11014–11019 (2002).
[CrossRef] [PubMed]

Rev. Sci. Instrum. (1)

J. Squier, M. Müller, “High resolution nonlinear microscopy: a review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum. 72, 2855–2867 (2001).
[CrossRef]

Scanning (1)

P.-C. Cheng, C.-K. Sun, B. L. Lin, F.-J. Kao, S.-W. Chu, “Biological multi-modality nonlinear spectromicroscopy: multiphoton fluorescence, second- and third-harmonic generation,” Scanning 23, 109–110 (2001), http://www.scanning.org .

Other (6)

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E. Baer, J. J. Cassidy, A. Hiltner, “Hierarchical structure of collagen and its relationship to the physical properties of tendon,” in Collagen: Biochemistry and Biomechanics, M. E. Nimni, ed. (CRC, Boca Raton, Fla., 1988), Vol. 2, pp. 177–199.

D. T. Poh, “Examination of refractive index of human epidermis in-vitro and in-vivo,” in Proceedings of the International Conference on Lasers ’96, V. J. Corcoran, T. A. Goldman, eds. (STS, McLean, Va., 1997), pp. 118–125.

D. J. Maitland, “Dynamic measurements of tissue birefringence: theory and experiments,” Ph.D. dissertation (Northwestern University, Evanston, Ill., 1995).

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R. W. Boyd, Nonlinear Optics (Academic, San Diego, Calif., 1992).

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Figures (8)

Fig. 1
Fig. 1

Schematic diagram of the experimental setup.

Fig. 2
Fig. 2

Parallel (resulting in enhanced second-harmonic signal) and antiparallel (resulting in cancellation of second-harmonic signal) orientation of neighboring collagen fibrils.

Fig. 3
Fig. 3

(a) SHG measurement in quartz. (b) Theoretical calculation of second-harmonic signal as a function of depth of focus (depth in sample increases with increasing z) generated in a quartz wave plate (z = 0 is the air-quartz interface) by beams focused with different Rayleigh ranges: top left, 50 μm; top right, 15 μm; bottom left, 5 μm; bottom right, 2 μm.

Fig. 4
Fig. 4

Intensity of second-harmonic signal as a function of depth in quartz near the top surface (increasing z corresponds to increasing depth in the sample) for the 20× objective and a 1-μm scan resolution (arbitrary origin).

Fig. 5
Fig. 5

Plot of the theoretically calculated peak signal (normalized to the value calculated for the 10× objective and the 100-mm collimating lens) versus the measured peak signal (normalized to the value measured for the 10× objective and the 100-mm collimating lens). Filled squares, data points; gray line (slope of 1), value expected for perfect agreement between theory and measurement.

Fig. 6
Fig. 6

Second-harmonic signal as a function of transverse position in a section of rat tail tendon obtained with a 1-μm scan resolution and (a) the 10× objective with the 100-mm collimating lens, (b) the 10× objective with the 200-mm collimating lens, (c) the 20× objective with the 50-mm collimating lens, (d) the 20× objective with the 100-mm collimating lens, (e) the 20× objective with the 200-mm collimating lens, (f) the 40× objective with the 50-mm collimating lens, (g) the 40× objective with the 100-mm collimating lens, and (h) the 40× objective with the 200-mm collimating lens. The sharp edges in the higher-resolution images are due to the finite pixel size; the data are not smoothed.

Fig. 7
Fig. 7

Polarization microscope image of a section of rat tail tendon.

Fig. 8
Fig. 8

Log-log plot of the frequency of occurrence versus second-harmonic signal intensity for the images shown in Fig. 5. The data are plotted by use of a different color for each of the objective-collimating lens combinations. The focal spot diameter calculated from the measurements in the quartz wave plate is given for each objective (refer to Table 3). In (a) the intensity data are not scaled (only the signal strength for a N.A. of 0.9, where significant undercollection of the signal occurs, has been corrected); in (b) the intensity data were scaled to compensate for the fact that intensity increases in inverse proportion to the square of the focal spot diameter. The intensity data were divided into 200 equally sized bins spanning the range 10-6–5 × 10-3 mV.

Tables (4)

Tables Icon

Table 1 Microscope Objectives Used in the Experiment

Tables Icon

Table 2 SHG in a Quartz Wave Plate for Several Objective and Collimating Lens Combinations

Tables Icon

Table 3 SHG in Quartz, Determining zR, w0, and |J|2

Tables Icon

Table 4 Lower Bound on the d-Tensor in Collagen

Equations (9)

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

P2ω=2|deffc|2πw02ω12n12n2c3ε0 P1ω2|J-zf, -zf+Lc, zR, Δkc|2.
Jzf, -zf+Lc, zR, Δkc=-zf-zf+LexpiΔkcz1+iz/zRdz.
P2ω=2|deffq|2πw02ω12n12n2c3ε0 P1ω2|J-zf, -zf+Lq, zR, Δkq|2,
P2ω  |J-zfmax, -zfmax+Lq, zR, Δkq|2w02.
deffc2=Vcollagen|Jzfmax, -zf+Lq, zR, Δkq|2Vquartz|J-zf, -zf+Lc, zR, Δkc|2 deffq2.
ISHG   3+20γ+40γ2-½ 1+6γ+8γ2cos2α+ 1+4γcos4α,
dXXXc2=1.05deffc2.
ISHG  +½ cos2πα+ cos4πα,
dXXX2=2.67deffq2,

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