Abstract

Collagen and myosin fibrils are endogenous harmonophores that both give rise to Second Harmonic Generation (SHG). By combining four polarization SHG images provided by a scanning microscope, we show that the orientation of the principal axis of the nonlinear susceptibility tensor χ (2) can be determined for each pixel of the image. The ratio ρ=χ33/χ15 of the principal components of χ (2) of collagen and myosin was obtained with the same method, and found within the range 1.6–1.8 and 0.5–0.6 respectively. The orientation of the principal axis of χ (2) is shown to be correlated to the orientation of the fibrils themselves. This provides a straightforward method, which we call Orientation Field-Second Harmonic Microscopy (OF-SHM), to reconstruct orientation fields of fibrils at various scales and resolutions in different biological systems (from muscle sarcomere to the whole embryo).

© 2008 Optical Society of America

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    [CrossRef]
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    [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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  29. M. H. Stromer, D. E. Goll, R. B. Young, R. M. Robson, and F. C. Parrish, "Ultrastructural features of skeletal muscle differentiation and development," Jr. J. Anim Sci. 38, 1111-1141 (1974).
  30. A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vie, D. Rouede, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, "Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second-harmonic generation and atomic force microscopy, Langmuir 20, 8165-8171 (2004).
    [CrossRef] [PubMed]
  31. O. P. Boryskina, Y. Le Grand, C. Odin, and V. Fleury, "The role of distribution and orientation of collagen fibers in tissue development: study by means of double imaging by two-photon excited fluorescence and second harmonic generation microscopy," Proc Europ. Microw. Assoc. 4, 255-259 (2008).
  32. M. L. Concha and R. J. Adams, "Oriented cell divisions and cellular morphogenesis in the zebrafish gastrula and neurula: a time-lapse analysis," Development 125, 983-994 (1998).
    [PubMed]

2008

C. Odin, Y. Le Grand, A. Renault, L. Gailhouste, and G. Baffet, "Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy," J. Microsc. 229, 32-38 (2008).
[CrossRef] [PubMed]

O. P. Boryskina, Y. Le Grand, C. Odin, and V. Fleury, "The role of distribution and orientation of collagen fibers in tissue development: study by means of double imaging by two-photon excited fluorescence and second harmonic generation microscopy," Proc Europ. Microw. Assoc. 4, 255-259 (2008).

X. Han, R. M. Burke, M. L. Zettel, P. Tang, and E. B. Brown, "Second harmonic properties of tumor collagen: determining the structural relationship between reactive stroma and healthy stroma," Opt. Express 16, 1846-1859 (2008), http://www.opticsinfobase.org/abstract.cfm?URI=oe-16-3-1846.
[CrossRef] [PubMed]

2007

2006

D. Debarre, A.-M. Pena, W. Supatto, T. Boulesteix, M. Strupler, M.-P. Sauviat, J.-L. Martin, M.-C. Schanne- Klein and E. Beaurepaire, "Second-and third-harmonic generation microscopies for the structural imaging of intact tissues, Med. Sci. 22, 845-850 (2006).

K. Kroy, "Elasticity, dynamics and relaxation in biopolymer networks," Curr. Opin. Colloin. Interface Sci. 11, 56-84 (2006).
[CrossRef]

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, "Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres," Biophys J. 90,693-703 (2006).
[CrossRef]

L. Gao, L. Jin, P. Xue, J. Xu, Y. Wang, H. Ma, and D. Chen, "Reconstruction of complementary images in second harmonic generation microscopy," Opt. Express 14, 4727-4735 (2006), http://www.opticsinfobase.org/abstract.cfm?URI=oe-14-11-4727.
[CrossRef] [PubMed]

2005

R. M. Williams, W. R. Zipfel, and W. W. Webb, "Interpreting Second-Harmonic Generation Images of Collagen I Fibrils," Biophys. J. 88, 1377-1386 (2005).
[CrossRef]

2004

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

T. Boulesteix, E. Beaurepaire, M. Sauviat, and M.-C. Schanne-Klein, "Second-harmonic microscopy of unstained living cardiac myocytes:measurements of sarcomere length with 20-nm accuracy," Opt. Lett. 29, 2031-2033 (2004). http://www.opticsinfobase.org/abstract.cfm?URI=ol-29-17-2031
[CrossRef] [PubMed]

S. Brasselet, V. Le Floch, F. Treussart, J.-F. Roch, J. Zyss, E. Botzung-Appert, and A. Ibanez, "In Situ Diagnostics of the Crystalline Nature of Single Organic Nanocrystals by Nonlinear Microscopy," Phys. Rev. Lett. 92, 207401- 207404 (2004).
[CrossRef] [PubMed]

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vie, D. Rouede, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, "Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second-harmonic generation and atomic force microscopy, Langmuir 20, 8165-8171 (2004).
[CrossRef] [PubMed]

P. J. Elbischger, H. Bischof, P. Regitnig, and G. A. Holzapfel, "Automatic analysis of collagen fiber orientation in the outermost layer of human arteries," Pattern Anal Applic 7, 269-284 (2004).

2003

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. USA. 100,7075-7080 (2003).
[CrossRef] [PubMed]

P. Fratzl, "Cellulose and collagen: from fibres to tissues," Curr. Op. Coll. Int. Sc. 8, 32-39 (2003).
[CrossRef]

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic : multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (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]

V. Le Floc'h, S. Brasselet, J.-F. Roch, and J. Zyss, "Monitoring of Orientation in Molecular Ensembles by Polarization Sensitive Nonlinear Microscopy," J. Phys. Chem. B 107, 12403-12410 (2003).
[CrossRef]

2002

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).

A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Nat. Acad. Sc. 20, 11014-11019 (2002).
[CrossRef]

2000

K. Konig, "Multiphoton microscopy in life sciences," J. Microsc. 200, 83-104 (2000).
[CrossRef] [PubMed]

1998

S. T. Jiang, "Contribution of Muscle Proteinases to Meat Tenderization," Proc. Natl. Sci. Council, ROC, Part B 22, 97-107 (1998).

M. L. Concha and R. J. Adams, "Oriented cell divisions and cellular morphogenesis in the zebrafish gastrula and neurula: a time-lapse analysis," Development 125, 983-994 (1998).
[PubMed]

1979

S. Roth and I. Freund, "Second harmonic generation in collagen," J. Chem. Phys. 70, 1637-1643(1979).
[CrossRef]

1974

M. H. Stromer, D. E. Goll, R. B. Young, R. M. Robson, and F. C. Parrish, "Ultrastructural features of skeletal muscle differentiation and development," Jr. J. Anim Sci. 38, 1111-1141 (1974).

1962

D. A. Kleinman, "Nonlinear Dielectric Polarization in Optical Media," Phys. Rev. 126, 1977-1979 (1962).
[CrossRef]

1951

H. Hamburger and H. L. Hamilton "A series of normal stages in the development of the chick embryo," J. Morphol. 88, 49-92 (1951).
[CrossRef]

Adams, R. J.

M. L. Concha and R. J. Adams, "Oriented cell divisions and cellular morphogenesis in the zebrafish gastrula and neurula: a time-lapse analysis," Development 125, 983-994 (1998).
[PubMed]

Baffet, G.

C. Odin, Y. Le Grand, A. Renault, L. Gailhouste, and G. Baffet, "Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy," J. Microsc. 229, 32-38 (2008).
[CrossRef] [PubMed]

Beaurepaire, E.

Bischof, H.

P. J. Elbischger, H. Bischof, P. Regitnig, and G. A. Holzapfel, "Automatic analysis of collagen fiber orientation in the outermost layer of human arteries," Pattern Anal Applic 7, 269-284 (2004).

Boryskina, O. P.

O. P. Boryskina, Y. Le Grand, C. Odin, and V. Fleury, "The role of distribution and orientation of collagen fibers in tissue development: study by means of double imaging by two-photon excited fluorescence and second harmonic generation microscopy," Proc Europ. Microw. Assoc. 4, 255-259 (2008).

Botzung-Appert, E.

S. Brasselet, V. Le Floch, F. Treussart, J.-F. Roch, J. Zyss, E. Botzung-Appert, and A. Ibanez, "In Situ Diagnostics of the Crystalline Nature of Single Organic Nanocrystals by Nonlinear Microscopy," Phys. Rev. Lett. 92, 207401- 207404 (2004).
[CrossRef] [PubMed]

Boulesteix, T.

Brasselet, S.

S. Brasselet, J. Zyss, "Nonlinear polarimetry of molecular crystals down to the nanoscale," C. R. Physique R. Adv. Cryst. Opt. 8, 165-179 (2007).

S. Brasselet, V. Le Floch, F. Treussart, J.-F. Roch, J. Zyss, E. Botzung-Appert, and A. Ibanez, "In Situ Diagnostics of the Crystalline Nature of Single Organic Nanocrystals by Nonlinear Microscopy," Phys. Rev. Lett. 92, 207401- 207404 (2004).
[CrossRef] [PubMed]

Brown, E. B.

Burke, R. M.

Campagnola, P. J.

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, "Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres," Biophys J. 90,693-703 (2006).
[CrossRef]

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]

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).

Chen, D.

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 |(2)/|(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914- 3922 (2004).
[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 |(2)/|(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914- 3922 (2004).
[CrossRef] [PubMed]

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 |(2)/|(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914- 3922 (2004).
[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. USA. 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 |(2)/|(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914- 3922 (2004).
[CrossRef] [PubMed]

Concha, M. L.

M. L. Concha and R. J. Adams, "Oriented cell divisions and cellular morphogenesis in the zebrafish gastrula and neurula: a time-lapse analysis," Development 125, 983-994 (1998).
[PubMed]

Elbischger, P. J.

P. J. Elbischger, H. Bischof, P. Regitnig, and G. A. Holzapfel, "Automatic analysis of collagen fiber orientation in the outermost layer of human arteries," Pattern Anal Applic 7, 269-284 (2004).

Fleury, V.

O. P. Boryskina, Y. Le Grand, C. Odin, and V. Fleury, "The role of distribution and orientation of collagen fibers in tissue development: study by means of double imaging by two-photon excited fluorescence and second harmonic generation microscopy," Proc Europ. Microw. Assoc. 4, 255-259 (2008).

Fratzl, P.

P. Fratzl, "Cellulose and collagen: from fibres to tissues," Curr. Op. Coll. Int. Sc. 8, 32-39 (2003).
[CrossRef]

Freund, I.

S. Roth and I. Freund, "Second harmonic generation in collagen," J. Chem. Phys. 70, 1637-1643(1979).
[CrossRef]

Gailhouste, L.

C. Odin, Y. Le Grand, A. Renault, L. Gailhouste, and G. Baffet, "Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy," J. Microsc. 229, 32-38 (2008).
[CrossRef] [PubMed]

Gao, L.

Goll, D. E.

M. H. Stromer, D. E. Goll, R. B. Young, R. M. Robson, and F. C. Parrish, "Ultrastructural features of skeletal muscle differentiation and development," Jr. J. Anim Sci. 38, 1111-1141 (1974).

Hamburger, H.

H. Hamburger and H. L. Hamilton "A series of normal stages in the development of the chick embryo," J. Morphol. 88, 49-92 (1951).
[CrossRef]

Hamilton, H. L.

H. Hamburger and H. L. Hamilton "A series of normal stages in the development of the chick embryo," J. Morphol. 88, 49-92 (1951).
[CrossRef]

Han, X.

Holzapfel, G. A.

P. J. Elbischger, H. Bischof, P. Regitnig, and G. A. Holzapfel, "Automatic analysis of collagen fiber orientation in the outermost layer of human arteries," Pattern Anal Applic 7, 269-284 (2004).

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. USA. 100,7075-7080 (2003).
[CrossRef] [PubMed]

Ibanez, A.

S. Brasselet, V. Le Floch, F. Treussart, J.-F. Roch, J. Zyss, E. Botzung-Appert, and A. Ibanez, "In Situ Diagnostics of the Crystalline Nature of Single Organic Nanocrystals by Nonlinear Microscopy," Phys. Rev. Lett. 92, 207401- 207404 (2004).
[CrossRef] [PubMed]

Jiang, S. T.

S. T. Jiang, "Contribution of Muscle Proteinases to Meat Tenderization," Proc. Natl. Sci. Council, ROC, Part B 22, 97-107 (1998).

Jin, L.

Kleinman, D. A.

D. A. Kleinman, "Nonlinear Dielectric Polarization in Optical Media," Phys. Rev. 126, 1977-1979 (1962).
[CrossRef]

Konig, K.

K. Konig, "Multiphoton microscopy in life sciences," J. Microsc. 200, 83-104 (2000).
[CrossRef] [PubMed]

Kroy, K.

K. Kroy, "Elasticity, dynamics and relaxation in biopolymer networks," Curr. Opin. Colloin. Interface Sci. 11, 56-84 (2006).
[CrossRef]

Le Floch, V.

S. Brasselet, V. Le Floch, F. Treussart, J.-F. Roch, J. Zyss, E. Botzung-Appert, and A. Ibanez, "In Situ Diagnostics of the Crystalline Nature of Single Organic Nanocrystals by Nonlinear Microscopy," Phys. Rev. Lett. 92, 207401- 207404 (2004).
[CrossRef] [PubMed]

Le Grand, Y.

C. Odin, Y. Le Grand, A. Renault, L. Gailhouste, and G. Baffet, "Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy," J. Microsc. 229, 32-38 (2008).
[CrossRef] [PubMed]

O. P. Boryskina, Y. Le Grand, C. Odin, and V. Fleury, "The role of distribution and orientation of collagen fibers in tissue development: study by means of double imaging by two-photon excited fluorescence and second harmonic generation microscopy," Proc Europ. Microw. Assoc. 4, 255-259 (2008).

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vie, D. Rouede, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, "Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second-harmonic generation and atomic force microscopy, Langmuir 20, 8165-8171 (2004).
[CrossRef] [PubMed]

Leray, A.

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vie, D. Rouede, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, "Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second-harmonic generation and atomic force microscopy, Langmuir 20, 8165-8171 (2004).
[CrossRef] [PubMed]

Leroy, L.

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vie, D. Rouede, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, "Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second-harmonic generation and atomic force microscopy, Langmuir 20, 8165-8171 (2004).
[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 |(2)/|(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914- 3922 (2004).
[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).
[CrossRef] [PubMed]

Ma, H.

Millard, A. C.

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, "Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres," Biophys J. 90,693-703 (2006).
[CrossRef]

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. USA. 100,7075-7080 (2003).
[CrossRef] [PubMed]

Odin, C.

C. Odin, Y. Le Grand, A. Renault, L. Gailhouste, and G. Baffet, "Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy," J. Microsc. 229, 32-38 (2008).
[CrossRef] [PubMed]

O. P. Boryskina, Y. Le Grand, C. Odin, and V. Fleury, "The role of distribution and orientation of collagen fibers in tissue development: study by means of double imaging by two-photon excited fluorescence and second harmonic generation microscopy," Proc Europ. Microw. Assoc. 4, 255-259 (2008).

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vie, D. Rouede, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, "Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second-harmonic generation and atomic force microscopy, Langmuir 20, 8165-8171 (2004).
[CrossRef] [PubMed]

Parrish, F. C.

M. H. Stromer, D. E. Goll, R. B. Young, R. M. Robson, and F. C. Parrish, "Ultrastructural features of skeletal muscle differentiation and development," Jr. J. Anim Sci. 38, 1111-1141 (1974).

Plotnikov, S. V.

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, "Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres," Biophys J. 90,693-703 (2006).
[CrossRef]

Recher, G.

Regitnig, P.

P. J. Elbischger, H. Bischof, P. Regitnig, and G. A. Holzapfel, "Automatic analysis of collagen fiber orientation in the outermost layer of human arteries," Pattern Anal Applic 7, 269-284 (2004).

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).

Renault, A.

C. Odin, Y. Le Grand, A. Renault, L. Gailhouste, and G. Baffet, "Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy," J. Microsc. 229, 32-38 (2008).
[CrossRef] [PubMed]

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vie, D. Rouede, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, "Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second-harmonic generation and atomic force microscopy, Langmuir 20, 8165-8171 (2004).
[CrossRef] [PubMed]

Robson, R. M.

M. H. Stromer, D. E. Goll, R. B. Young, R. M. Robson, and F. C. Parrish, "Ultrastructural features of skeletal muscle differentiation and development," Jr. J. Anim Sci. 38, 1111-1141 (1974).

Roch, J.-F.

S. Brasselet, V. Le Floch, F. Treussart, J.-F. Roch, J. Zyss, E. Botzung-Appert, and A. Ibanez, "In Situ Diagnostics of the Crystalline Nature of Single Organic Nanocrystals by Nonlinear Microscopy," Phys. Rev. Lett. 92, 207401- 207404 (2004).
[CrossRef] [PubMed]

Roth, S.

S. Roth and I. Freund, "Second harmonic generation in collagen," J. Chem. Phys. 70, 1637-1643(1979).
[CrossRef]

Rouede, D.

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).

Sauviat, M.

Schanne-Klein, M.-C.

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).

Stromer, M. H.

M. H. Stromer, D. E. Goll, R. B. Young, R. M. Robson, and F. C. Parrish, "Ultrastructural features of skeletal muscle differentiation and development," Jr. J. Anim Sci. 38, 1111-1141 (1974).

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 |(2)/|(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914- 3922 (2004).
[CrossRef] [PubMed]

Tang, P.

Tiaho, F.

Treussart, F.

S. Brasselet, V. Le Floch, F. Treussart, J.-F. Roch, J. Zyss, E. Botzung-Appert, and A. Ibanez, "In Situ Diagnostics of the Crystalline Nature of Single Organic Nanocrystals by Nonlinear Microscopy," Phys. Rev. Lett. 92, 207401- 207404 (2004).
[CrossRef] [PubMed]

Tromberg, B. J.

A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Nat. Acad. Sc. 20, 11014-11019 (2002).
[CrossRef]

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 |(2)/|(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914- 3922 (2004).
[CrossRef] [PubMed]

Wang, Y.

Webb, W. W.

R. M. Williams, W. R. Zipfel, and W. W. Webb, "Interpreting Second-Harmonic Generation Images of Collagen I Fibrils," Biophys. J. 88, 1377-1386 (2005).
[CrossRef]

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic : multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

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. USA. 100,7075-7080 (2003).
[CrossRef] [PubMed]

Williams, R. M.

R. M. Williams, W. R. Zipfel, and W. W. Webb, "Interpreting Second-Harmonic Generation Images of Collagen I Fibrils," Biophys. J. 88, 1377-1386 (2005).
[CrossRef]

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. USA. 100,7075-7080 (2003).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic : multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

Xu, J.

Xue, P.

Yeh, A.

A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Nat. Acad. Sc. 20, 11014-11019 (2002).
[CrossRef]

Young, R. B.

M. H. Stromer, D. E. Goll, R. B. Young, R. M. Robson, and F. C. Parrish, "Ultrastructural features of skeletal muscle differentiation and development," Jr. J. Anim Sci. 38, 1111-1141 (1974).

Zettel, M. L.

Zipfel, W. R.

R. M. Williams, W. R. Zipfel, and W. W. Webb, "Interpreting Second-Harmonic Generation Images of Collagen I Fibrils," Biophys. J. 88, 1377-1386 (2005).
[CrossRef]

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic : multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

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. USA. 100,7075-7080 (2003).
[CrossRef] [PubMed]

Zoumi, A.

A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Nat. Acad. Sc. 20, 11014-11019 (2002).
[CrossRef]

Zyss, J.

S. Brasselet, J. Zyss, "Nonlinear polarimetry of molecular crystals down to the nanoscale," C. R. Physique R. Adv. Cryst. Opt. 8, 165-179 (2007).

S. Brasselet, V. Le Floch, F. Treussart, J.-F. Roch, J. Zyss, E. Botzung-Appert, and A. Ibanez, "In Situ Diagnostics of the Crystalline Nature of Single Organic Nanocrystals by Nonlinear Microscopy," Phys. Rev. Lett. 92, 207401- 207404 (2004).
[CrossRef] [PubMed]

Biophys J.

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, "Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres," Biophys J. 90,693-703 (2006).
[CrossRef]

Biophys. J.

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).

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

R. M. Williams, W. R. Zipfel, and W. W. Webb, "Interpreting Second-Harmonic Generation Images of Collagen I Fibrils," Biophys. J. 88, 1377-1386 (2005).
[CrossRef]

C. R. Physique R. Adv. Cryst. Opt.

S. Brasselet, J. Zyss, "Nonlinear polarimetry of molecular crystals down to the nanoscale," C. R. Physique R. Adv. Cryst. Opt. 8, 165-179 (2007).

Curr. Op. Coll. Int. Sc.

P. Fratzl, "Cellulose and collagen: from fibres to tissues," Curr. Op. Coll. Int. Sc. 8, 32-39 (2003).
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Curr. Opin. Colloin. Interface Sci.

K. Kroy, "Elasticity, dynamics and relaxation in biopolymer networks," Curr. Opin. Colloin. Interface Sci. 11, 56-84 (2006).
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S. Roth and I. Freund, "Second harmonic generation in collagen," J. Chem. Phys. 70, 1637-1643(1979).
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J. Microsc.

K. Konig, "Multiphoton microscopy in life sciences," J. Microsc. 200, 83-104 (2000).
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C. Odin, Y. Le Grand, A. Renault, L. Gailhouste, and G. Baffet, "Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy," J. Microsc. 229, 32-38 (2008).
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V. Le Floc'h, S. Brasselet, J.-F. Roch, and J. Zyss, "Monitoring of Orientation in Molecular Ensembles by Polarization Sensitive Nonlinear Microscopy," J. Phys. Chem. B 107, 12403-12410 (2003).
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M. H. Stromer, D. E. Goll, R. B. Young, R. M. Robson, and F. C. Parrish, "Ultrastructural features of skeletal muscle differentiation and development," Jr. J. Anim Sci. 38, 1111-1141 (1974).

Langmuir

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vie, D. Rouede, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, "Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second-harmonic generation and atomic force microscopy, Langmuir 20, 8165-8171 (2004).
[CrossRef] [PubMed]

Med. Sci.

D. Debarre, A.-M. Pena, W. Supatto, T. Boulesteix, M. Strupler, M.-P. Sauviat, J.-L. Martin, M.-C. Schanne- Klein and E. Beaurepaire, "Second-and third-harmonic generation microscopies for the structural imaging of intact tissues, Med. Sci. 22, 845-850 (2006).

Nat. Biotechnol.

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic : multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (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]

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P. J. Elbischger, H. Bischof, P. Regitnig, and G. A. Holzapfel, "Automatic analysis of collagen fiber orientation in the outermost layer of human arteries," Pattern Anal Applic 7, 269-284 (2004).

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S. Brasselet, V. Le Floch, F. Treussart, J.-F. Roch, J. Zyss, E. Botzung-Appert, and A. Ibanez, "In Situ Diagnostics of the Crystalline Nature of Single Organic Nanocrystals by Nonlinear Microscopy," Phys. Rev. Lett. 92, 207401- 207404 (2004).
[CrossRef] [PubMed]

Proc Europ. Microw. Assoc.

O. P. Boryskina, Y. Le Grand, C. Odin, and V. Fleury, "The role of distribution and orientation of collagen fibers in tissue development: study by means of double imaging by two-photon excited fluorescence and second harmonic generation microscopy," Proc Europ. Microw. Assoc. 4, 255-259 (2008).

Proc. Nat. Acad. Sc.

A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Nat. Acad. Sc. 20, 11014-11019 (2002).
[CrossRef]

Proc. Natl. Acad. Sci. USA.

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. USA. 100,7075-7080 (2003).
[CrossRef] [PubMed]

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Figures (9)

Fig. 1.
Fig. 1.

(color online) : Colocalization of TPEF and SHG signals on veal fresh muscle with a RGB color coding. Full scale 1024×1024 images, 60xW-NA1.2 objective : (a) TPEF (red); (b) Isotropic SHG from Myosin (green); (c) TPEF+SHG (red and green) RGB image. Scale bar : 40µm. (d-e-f) Zoom corresponding to the white rectangle in image (c), scale bar : 20µm. (g) Sections of the TPEF and SHG images along the bundle of myofibrils; (h) Spatial Fourier transforms of 65µm sections.

Fig. 2.
Fig. 2.

(color online) : (a) Schematic of a fibril carrying the nonlinear susceptibility χ (2) in the laboratory frame : laser polarization and C symmetry axis Zχ angles with respect to X axis are ψ and ϕ respectively ; (c) Two interpretations of the polar angle θ used to calculate ρ in Eq.(10) for C harmonophore structures. (c) SHG and TPEF experimental set-up, with laser polarization control. DM=Dichroic mirror, PMT=Photomultiplier Tube.

Fig. 3.
Fig. 3.

SHG of sheep tendon collagen (60xW-NA1.2 objective, 512×512 images, full scale 235µm). (a1–a4) Acquisition with four polarizations indicated by the double white arrows; (b) isotropic image; (c) Orientation field represented by bars directed along the symmetry axis of the nonlinear susceptibility (one bar for each bloc of 16×16 pixels); (d) Masks selecting different fibrils; (e) Correlation between the apparent orientation of the fibrils (abscissa) and the orientation of the principal axis of the SHG nonlinear susceptibility (ordinate).

Fig. 4.
Fig. 4.

SHG of sheep tendon collagen (10x-NA0.25 objective, 256×256 images, full scale 625µm). (a1–a4) 4 polarization images for the reference orientation. (b) Orientation field of the a-image by blocks of 16×16 pixels. (c1–c4) 4 polarization images of the sample that was physically rotated by 20°; (d) Orientation Field of the c-image; (e) Histograms of the orientations. For clarity, the different histograms were vertically shifted : (Top) histograms for all the pixels of the image; (Middle and Bottom) Histograms of the 80×80 ROI at the top left and bottom right corners of the image respectively; The histograms were normalized with a weight corresponding to the size of the ROI. Continuous lines are gaussian fits.

Fig. 5.
Fig. 5.

SHG images from myosin of the muscle of the back of a 7 days chicken embryo (10x-NA0.25 objective, 768×768 images, full scale 938µm). Scale bar : 100µm. The legends are similar to Fig. 3.

Fig. 6.
Fig. 6.

(Color online) TPEF, myosin SHG and orientation field of veal muscle at two scales. First raw: 10x-NA0.25 objective, 400×400 images, scale bar 100µm. (a) TPEF; (b) SHG; (c) Orientation field superimposed on the isotropic SHG image. Second raw : 60xW-NA1.2 objective, 1024×1024 images, full scale 235µm; (d1–4) 4 polarization images; (e) Orientation Field; (f) Detail of the orientation field with superimposed isotropic SHG image.

Fig. 7.
Fig. 7.

Relationship between |V|/U and ρ from Eq.(12).(a) Histogram of |V|/U for sheep tendon collagen obtained from a 512×512 image acquired with an 60xW-NA1.2 objective; (b) Theoretical relationship Eq.(12) between ρ and |V|/U when 0<ρ<1 or ρ>1; (c) Histogram of ρ values corresponding to the |V|/U values of Fig. 7(a).

Fig. 8.
Fig. 8.

SHG images of the skin of a 9 days (HH35) chicken embryo (10x-NA0.25 objective).(a) Full scale SHG image (1024×1024) ;(b) Zoom of the ROI delineated by the white rectangle (512×512 image) ;(c) Orientation field. Scale bars : 100µm.

Fig. 9.
Fig. 9.

(color online) Head of a HH8 Chicken Embryo imaged with a 10x-NA0.25 objective (700×700 image, 940×940µm2). (a) TPEF (Red) ;(b) Collagen SHG (green) ;(c) TPEF+SHG ;(d) Orientation field ;(e) Masks delineating the border of the embryo ;(f) Correlation between the SHG and masks orientations, corrected by 90°.

Tables (1)

Tables Icon

Table 1. Mean values of ρ for collagen and myosin. <ρ> and σρ are respectively the mean and standard deviation of ρ calculated directly from the raw data with usual formula. The polar angle θ ¯ and its incertitude Δθ were calculated as explained in the text.

Equations (15)

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

I ( ϕ , ψ ) = U + V cos ( 2 ψ 2 ϕ ) + W cos ( 4 ψ 4 ϕ )
U = I 8 ( 3 ρ 2 + 2 ρ + 7 )
V = I 2 ( ρ 2 1 )
W = I 8 ( ρ 2 2 ρ 3 )
I 02 ( ϕ ) = [ I 0 ( ϕ ) I 2 ( ϕ ) ] 2 = V cos ( 2 ϕ )
I 31 ( ϕ ) = [ I 3 ( ϕ ) I 1 ( ϕ ) ] 2 = V sin ( 2 ϕ )
at an 2 ( s , c ) = arctan ( s c ) if c > 0
at an 2 ( s , c ) = π + arctan ( s c ) if c < 0 and s > 0
at an 2 ( s , c ) = π + arctan ( s c ) if c < 0 and s < 0
if V > 0 , 2 ϕ = at an 2 ( I 31 , I 02 )
if V < 0 , 2 ϕ = at an 2 ( I 31 , I 02 )
ϕ M = 1 2 at an 2 ( Σ α , β M sin ( ϕ α β ) Σ α , β M cos ( ϕ α β ) )
ρ = 2 tan 2 θ
V U = 4 [ ρ 2 1 ] [ 3 ρ 2 + 2 ρ + 7 ]
ρ = r + 2 4 ( 1 + r ) 5 r 2 4 3 r

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