We use nonlinear optical microscopy combining Second Harmonic Generation (SHG) microscopy and Two-Photon Excited Fluorescence (2PEF) signals to characterize collagen lyotropic liquid crystals. We show that SHG signals provide highly contrasted images of the three-dimensional texture of cholesteric patterns with submicrometer lateral resolution. Moreover, simultaneous recording of the 2PEF signal enables in situ quantitative mapping of the molecular concentration and its correlation with the observed textures. We apply this technique to the characterization of biomimetic textures obtained in concentrated collagen liquid solutions. We successfully image biologically relevant organizations that are similar to the collagen organization found as a stabilized state in compact bones.

© 2010 OSA

1. M. Schadt, “Liquid crystal materials and liquid crystal displays,” Annu. Rev. Mater. Sci. 27(1), 305–379 (1997). [CrossRef]
2. K. Amundson, A. van Blaaderen, and P. Wiltzius, “Morphology and electro-optic properties of polymer-dispersed liquid-crystal films,” Phys. Rev. E Stat. Phys. Plasmas Fluids Relat. Interdiscip. Topics 55(2), 1646–1654 (1997). [CrossRef]
3. G. A. Held, L. L. Kosbar, I. Dierking, A. C. Lowe, G. Grinstein, V. Lee, and R. D. Miller, “Confocal Microscopy Study of Texture Transitions in a Polymer Stabilized Cholesteric Liquid Crystal,” Phys. Rev. Lett. 79(18), 3443–3446 (1997). [CrossRef]
4. M. Ofuji, Y. Takano, Y. Houkawa, Y. Takanishi, K. Ishikawa, H. Takezoe, T. Mori, M. Goh, S. Guo, and K. Akagi, “Microscopic orientational order of polymer chains in helical polyacetylene thin films studied by confocal laser raman microscopy,” Jpn. J. Appl. Phys. 45(No. 3A), 1710–1713 (2006). [CrossRef]
5. I. C. Khoo, “Nonlinear optics of liquid crystalline materials,” Phys. Rep. 471(5-6), 221–267 (2009). [CrossRef]
6. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990). [CrossRef] [PubMed]
7. R. S. Pillai, M. Oh-E, H. Yokoyama, G. J. Brakenhoff, and M. Müller, “Imaging colloidal particle induced topological defects in a nematic liquid crystal using third harmonic generation microscopy,” Opt. Express 14(26), 12976–12983 (2006). [CrossRef] [PubMed]
8. D. Yelin, Y. Silberberg, Y. Barad, and J. S. Patel, “Depth-resolved imaging of nematic liquid crystals by third-harmonic microscopy,” Appl. Phys. Lett. 74(21), 3107–3109 (1999). [CrossRef]
9. D. Yelin, Y. Silberberg, Y. Barad, and J. S. Patel, “Phase-matched third-harmonic generation in a nematic liquid crystal cell,” Phys. Rev. Lett. 82(15), 3046–3049 (1999). [CrossRef]
10. L. Moreaux, O. Sandre, and J. Mertz, “Membrane imaging by second-harmonic generation microscopy,” J. Opt. Soc. Am. B 17(10), 1685–1694 (2000). [CrossRef]
11. P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-modulated second harmonic generation in collagen,” Biophys. J. 82(6), 3330–3342 (2002). [CrossRef] [PubMed]
12. P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-Dimensional High-Resolution Second-Harmonic Generation Imaging of Endogenous Structural Proteins in Biological Tissues,” Biophys. J. 82(1 Pt 1), 493–508 (2002). [CrossRef]
13. R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen fibrils,” Biophys. J. 88(2), 1377–1386 (2005). [CrossRef]
14. M. Strupler, A.-M. Pena, M. Hernest, P.-L. Tharaux, J.-L. Martin, E. Beaurepaire, and M.-C. Schanne-Klein, “Second harmonic imaging and scoring of collagen in fibrotic tissues,” Opt. Express 15(7), 4054–4065 (2007). [CrossRef] [PubMed]
15. O. G. Morales-Saavedra, M. Bulat, S. Rauch, and G. Heppke, “Domain structure studies in phases of bent-shaped molecules by spatially resolved second harmonic microscopy,” Mol. Cryst. Liq. Cryst. (Phila. Pa.) 413, 2743–2752 (2004). [CrossRef]
16. H. Kim, T. K. Lim, S. T. Shin, C. K. Lee, F. Araoka, M. Ofuji, Y. Takanishi, and H. Takezoe, “Coexistence of polar and nonpolar domains and their photocontrol in the B-7 phase of a bent-core liquid crystal containing azo dyes,” Phys. Rev. E 69, - (2004).
17. A. Deniset-Besseau, J. Duboisset, E. Benichou, F. Hache, P.-F. Brevet, and M.-C. Schanne-Klein, “Measurement of the second-order hyperpolarizability of the collagen triple helix and determination of its physical origin,” J. Phys. Chem. B 113(40), 13437–13445 (2009). [CrossRef] [PubMed]
18. F. Gobeaux, G. Mosser, A. Anglo, P. Panine, P. Davidson, M.-M. Giraud-Guille, and E. Belamie, “Fibrillogenesis in dense collagen solutions: a physicochemical study,” J. Mol. Biol. 376(5), 1509–1522 (2008). [CrossRef] [PubMed]
19. M.-M. Giraud-Guille, G. Mosser, and E. Belamie, “Liquid crystallinity in collagen systems in vitro and in vivo,” Curr. Opin. Colloid In. 13(4), 303–313 (2008). [CrossRef]
20. D. J. S. Hulmes, “Building Collagen Molecules, Fibrils, and Suprafibrillar Structures,” J. Struct. Biol. 137(1-2), 2–10 (2002). [CrossRef] [PubMed]
21. F. Gobeaux, E. Belamie, G. Mosser, P. Davidson, P. Panine, and M.-M. Giraud-Guille, “Cooperative ordering of collagen triple helices in the dense state,” Langmuir 23(11), 6411–6417 (2007). [CrossRef] [PubMed]
22. G. Mosser, A. Anglo, C. Helary, Y. Bouligand, and M.-M. Giraud-Guille, “Dense tissue-like collagen matrices formed in cell-free conditions,” Matrix Biol. 25(1), 3–13 (2006). [CrossRef]
23. P. De Sa Peixoto, A. Deniset-Besseau, A. Anglo, C. Illoul, M.-C. Schanne-Klein, and G. Mosser, (in preparation).
24. I. Bergman and R. Loxley, “2 Improved and Simplified Methods for Spectrophotometric Determination of Hydroxyproline,” Anal. Chem. 35, 1961-& (1963).
25. F. Gobeaux, E. Belamie, G. Mosser, P. Davidson, and S. Asnacios, “Power law rheology and strain-induced yielding in acidic solutions of type I-collagen,” submitted.
26. S. Roth and I. Freund, “Second harmonic generation in collagen,” J. Chem. Phys. 70(4), 1637–1643 (1979). [CrossRef]
27. N. Olivier and E. Beaurepaire, “Third-harmonic generation microscopy with focus-engineered beams: a numerical study,” Opt. Express 16(19), 14703–14715 (2008). [CrossRef] [PubMed]
28. M.-M. Giraud-Guille, “Twisted Plywood Architecture of Collagen Fibrils in Human Compact-Bone Osteons,” Calcif. Tissue Int. 42(3), 167–180 (1988). [CrossRef] [PubMed]

Annu. Rev. Mater. Sci.

• M. Schadt, “Liquid crystal materials and liquid crystal displays,” Annu. Rev. Mater. Sci. 27(1), 305–379 (1997). [CrossRef]

Appl. Phys. Lett.

• D. Yelin, Y. Silberberg, Y. Barad, and J. S. Patel, “Depth-resolved imaging of nematic liquid crystals by third-harmonic microscopy,” Appl. Phys. Lett. 74(21), 3107–3109 (1999). [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(6), 3330–3342 (2002). [CrossRef] [PubMed]
• P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-Dimensional High-Resolution Second-Harmonic Generation Imaging of Endogenous Structural Proteins in Biological Tissues,” Biophys. J. 82(1 Pt 1), 493–508 (2002). [CrossRef]
• R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen fibrils,” Biophys. J. 88(2), 1377–1386 (2005). [CrossRef]

Calcif. Tissue Int.

• M.-M. Giraud-Guille, “Twisted Plywood Architecture of Collagen Fibrils in Human Compact-Bone Osteons,” Calcif. Tissue Int. 42(3), 167–180 (1988). [CrossRef] [PubMed]

Curr. Opin. Colloid In.

• M.-M. Giraud-Guille, G. Mosser, and E. Belamie, “Liquid crystallinity in collagen systems in vitro and in vivo,” Curr. Opin. Colloid In. 13(4), 303–313 (2008). [CrossRef]

J. Chem. Phys.

• S. Roth and I. Freund, “Second harmonic generation in collagen,” J. Chem. Phys. 70(4), 1637–1643 (1979). [CrossRef]

J. Mol. Biol.

• F. Gobeaux, G. Mosser, A. Anglo, P. Panine, P. Davidson, M.-M. Giraud-Guille, and E. Belamie, “Fibrillogenesis in dense collagen solutions: a physicochemical study,” J. Mol. Biol. 376(5), 1509–1522 (2008). [CrossRef] [PubMed]

J. Opt. Soc. Am. B

• L. Moreaux, O. Sandre, and J. Mertz, “Membrane imaging by second-harmonic generation microscopy,” J. Opt. Soc. Am. B 17(10), 1685–1694 (2000). [CrossRef]

J. Phys. Chem. B

• A. Deniset-Besseau, J. Duboisset, E. Benichou, F. Hache, P.-F. Brevet, and M.-C. Schanne-Klein, “Measurement of the second-order hyperpolarizability of the collagen triple helix and determination of its physical origin,” J. Phys. Chem. B 113(40), 13437–13445 (2009). [CrossRef] [PubMed]

J. Struct. Biol.

• D. J. S. Hulmes, “Building Collagen Molecules, Fibrils, and Suprafibrillar Structures,” J. Struct. Biol. 137(1-2), 2–10 (2002). [CrossRef] [PubMed]

Jpn. J. Appl. Phys.

• M. Ofuji, Y. Takano, Y. Houkawa, Y. Takanishi, K. Ishikawa, H. Takezoe, T. Mori, M. Goh, S. Guo, and K. Akagi, “Microscopic orientational order of polymer chains in helical polyacetylene thin films studied by confocal laser raman microscopy,” Jpn. J. Appl. Phys. 45(No. 3A), 1710–1713 (2006). [CrossRef]

Langmuir

• F. Gobeaux, E. Belamie, G. Mosser, P. Davidson, P. Panine, and M.-M. Giraud-Guille, “Cooperative ordering of collagen triple helices in the dense state,” Langmuir 23(11), 6411–6417 (2007). [CrossRef] [PubMed]

Matrix Biol.

• G. Mosser, A. Anglo, C. Helary, Y. Bouligand, and M.-M. Giraud-Guille, “Dense tissue-like collagen matrices formed in cell-free conditions,” Matrix Biol. 25(1), 3–13 (2006). [CrossRef]

Mol. Cryst. Liq. Cryst. (Phila. Pa.)

• O. G. Morales-Saavedra, M. Bulat, S. Rauch, and G. Heppke, “Domain structure studies in phases of bent-shaped molecules by spatially resolved second harmonic microscopy,” Mol. Cryst. Liq. Cryst. (Phila. Pa.) 413, 2743–2752 (2004). [CrossRef]

Opt. Express

• M. Strupler, A.-M. Pena, M. Hernest, P.-L. Tharaux, J.-L. Martin, E. Beaurepaire, and M.-C. Schanne-Klein, “Second harmonic imaging and scoring of collagen in fibrotic tissues,” Opt. Express 15(7), 4054–4065 (2007). [CrossRef] [PubMed]
• R. S. Pillai, M. Oh-E, H. Yokoyama, G. J. Brakenhoff, and M. Müller, “Imaging colloidal particle induced topological defects in a nematic liquid crystal using third harmonic generation microscopy,” Opt. Express 14(26), 12976–12983 (2006). [CrossRef] [PubMed]
• N. Olivier and E. Beaurepaire, “Third-harmonic generation microscopy with focus-engineered beams: a numerical study,” Opt. Express 16(19), 14703–14715 (2008). [CrossRef] [PubMed]

Phys. Rep.

• I. C. Khoo, “Nonlinear optics of liquid crystalline materials,” Phys. Rep. 471(5-6), 221–267 (2009). [CrossRef]

Phys. Rev. E Stat. Phys. Plasmas Fluids Relat. Interdiscip. Topics

• K. Amundson, A. van Blaaderen, and P. Wiltzius, “Morphology and electro-optic properties of polymer-dispersed liquid-crystal films,” Phys. Rev. E Stat. Phys. Plasmas Fluids Relat. Interdiscip. Topics 55(2), 1646–1654 (1997). [CrossRef]

Phys. Rev. Lett.

• G. A. Held, L. L. Kosbar, I. Dierking, A. C. Lowe, G. Grinstein, V. Lee, and R. D. Miller, “Confocal Microscopy Study of Texture Transitions in a Polymer Stabilized Cholesteric Liquid Crystal,” Phys. Rev. Lett. 79(18), 3443–3446 (1997). [CrossRef]
• D. Yelin, Y. Silberberg, Y. Barad, and J. S. Patel, “Phase-matched third-harmonic generation in a nematic liquid crystal cell,” Phys. Rev. Lett. 82(15), 3046–3049 (1999). [CrossRef]

Science

• W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990). [CrossRef] [PubMed]

Other

• H. Kim, T. K. Lim, S. T. Shin, C. K. Lee, F. Araoka, M. Ofuji, Y. Takanishi, and H. Takezoe, “Coexistence of polar and nonpolar domains and their photocontrol in the B-7 phase of a bent-core liquid crystal containing azo dyes,” Phys. Rev. E 69, - (2004).
• P. De Sa Peixoto, A. Deniset-Besseau, A. Anglo, C. Illoul, M.-C. Schanne-Klein, and G. Mosser, (in preparation).
• I. Bergman and R. Loxley, “2 Improved and Simplified Methods for Spectrophotometric Determination of Hydroxyproline,” Anal. Chem. 35, 1961-& (1963).
• F. Gobeaux, E. Belamie, G. Mosser, P. Davidson, and S. Asnacios, “Power law rheology and strain-induced yielding in acidic solutions of type I-collagen,” submitted.

Click here to see a list of articles that cite this paper

Multimedia

Related Journal Articles

• Fourier analysis of the femtosecond hyper-Rayleigh scattering signal from ionic fluorescent hemicyanine dyes (JOSAB)
• Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy (OL)
• Harmonic microscopy of isotropic and anisotropic microstructure of the human cornea (OE)
• Three dimensional optical manipulation and structural imaging of soft materials by use of laser tweezers and multimodal nonlinear microscopy (OE)
• In situ three-dimensional monitoring of collagen fibrillogenesis using SHG microscopy (BOE)

Related Conference Papers

• Two-photon Absorption Studies of Polymethine and Squaraine Dyes with Analogical Structures
• Nonlinear Absorption Spectrum in Perylene Derivatives
• Observation of Quantum Interference in an Organic Solid
• Nonlinear Absorption Spectrum in Perylene Derivatives
• Second-harmonic generation in nonpolar chiral materials: relationship between molecular and macroscopic properties
• Second-harmonic generation in nonpolar chiral materials: relationship between molecular and macroscopic properties