Advertisement

Second-Harmonic Imaging of Collagen

  • Guy Cox
  • Eleanor Kable
Part of the Methods in Molecular Biology™ book series (MIMB, volume 319)

Abstract

Molecules that have no center of symmetry are able to convert light to its second harmonic, at twice the frequency and half the wavelength. This only happens with any efficiency at very high light intensities such as are given by a pulsed laser, and because the efficiency of the process depends on the square of the intensity, it will be focal plane selective in exactly the same way as two-photon excitation of fluorescence. Because of its unusual molecular structure and its high degree of crystallinity, collagen is, by far, the strongest source of second harmonics in animal tissue. Because collagen is also the most important structural protein in the mammalian body, this provides a very useful imaging tool for studying its distribution. No energy is lost in second-harmonic imaging, so the image will not fade, and because it is at a shorter wavelength than can be excited by two-photon fluorescence, it can be separated easily from multiple fluorescent probes. It is already proving useful in imaging collagen with high sensitivity in various tissues, including cirrhotic liver, normal and carious teeth, and surgical repair of tendons.

Key Words

Collagen second harmonic structural proteins biological imaging 3D imaging non-linear microscopy matrix 

References

  1. 1.
    Franken, P. A., Hill, A. E., Peters, C. W., and Weinreich, G. (1961) Generation of optical harmonics. Phys. Rev. Lett. 7, 118–119.CrossRefGoogle Scholar
  2. 2.
    Gauderon, R., Lukins, P. B., and Sheppard, C. J. R. (2001) Simultaneous multichannel nonlinear imaging: combined two-photon excited fluorescence and second harmonic generation microscopy. Micron 32, 685–689.PubMedCrossRefGoogle Scholar
  3. 3.
    Hellwarth, R. and Christensen, P. (1974) Nonlinear microscopic examination of structure in polycrystalline ZnSe. Opt. Commun. 12, 318–322.CrossRefGoogle Scholar
  4. 4.
    Gannaway, J. N. and Sheppard, C. J. R. (1978) Second harmonic imaging in the scanning optical microscope. Opt. Quant. Electron. 10, 435.CrossRefGoogle Scholar
  5. 5.
    Campagnola, P., Clark, H. A., Mohler, W. A., Lewis, A., and Loew, L. M. (2001) Second-harmonic imaging of living cells. J. Biomed. Opt. 6, 277–286.PubMedCrossRefGoogle Scholar
  6. 6.
    Campagnola, P. J., Millard, A. C., Terasaki, M., Hoppe, P. E., Malone, C. J., and Mohler, W. A. (2002) Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. Biophys. J. 81, 493–508.CrossRefGoogle Scholar
  7. 7.
    Mertz, J. and Moreaux, L. (2001) Multi-harmonic light microscopy: theory and applications to membrane imaging, in Multiphoton Microscopy in the Biomedical Sciences (A. Periasamy and P.T.C. So, eds.), Proceedings of SPIE 4262, 9–17.Google Scholar
  8. 8.
    Moreaux, L., Sandre, O., Charpak, S., Blanchard-Desce, M., and Mertz, J. (2001) Coherent scattering in multi-harmonic microscopy. Biophys. J. 80, 1568–1574.PubMedCrossRefGoogle Scholar
  9. 9.
    Lodish, H., Berk, A., Lipursky, S. L., Matsudaira, P., Baltimore, D., and Darrell, J. (2000) Molecular Cell Biology, 4th ed., W. H. Freeman, New York.Google Scholar
  10. 10.
    Roth, S. and Freund, I. (1981) Optical second-harmonic scattering in rat-tail tendon. Biopolymers 20, 1271–1290.PubMedCrossRefGoogle Scholar
  11. 11.
    Georgiou, E., Theodossiou, T., Hovhannisya, V., Politopoulos, K., Rapti, G. S., and Yova, D. (2000) Second and third optical harmonic generation in type I collagen, by nanosecond laser irradiation, over a broad spectral region. Opt. Commun. 176, 253–260.CrossRefGoogle Scholar
  12. 12.
    Cox, G., Kable, E., Jones, A., Fraser, I., Manconi, F., and Gorrell, M. (2002) 3-dimensional imaging of collagen using second harmonic generation. J. Struct. Biol. 141, 53–62.CrossRefGoogle Scholar
  13. 13.
    Freund, I., Deutsch, M., and Sprecher, A. (1986) Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon. Biophys. J. 50, 693–712.PubMedCrossRefGoogle Scholar
  14. 14.
    Kim, B. M., Eichler, J., Reiser, K. M., Rubenchik, A. M., and Da Silva, L. B. (2000) Collagen structure and nonlinear susceptibility: effects of heat, glycation, and enzymatic cleavage on second harmonic signal intensity. Lasers Surg. Med. 27, 329–335.PubMedCrossRefGoogle Scholar
  15. 15.
    Deng, X., Williams, E. D., Thompson, E. W., Gan, X., and Gu, M. (2002) Second harmonic generation from biological tissues: effect of excitation wavelength. Scanning 24, 175–178.PubMedCrossRefGoogle Scholar
  16. 16.
    Cox, G. C., Manconi, F., and Kable, E. (2002) Second harmonic imaging of collagen in mammalian tissue. Proc. SPIE 4620, 148–156.CrossRefGoogle Scholar
  17. 17.
    Blab, G. A., Lommerse, P. H. M., Cognet, L., Harms, G. S., and Schmidt, T. (2001) Two-photon excitation action cross-sections of the autofluorescent proteins. Chem. Phys. Lett. 350, 71–77.CrossRefGoogle Scholar
  18. 18.
    Ji-Xin Cheng, Kevin Jia, Y., Gengfeng Zheng, and Sunney Xie, X. (2002) Laserscanning coherent anti-Stokes Raman scattering microscopy and applications to cell biology. Biophys. J. 83, 502–509.CrossRefGoogle Scholar
  19. 19.
    Cox, G., Kable, E., Sheppard, C. J. R., and Xu, P. (2002) Resolution of second harmonic generation microscopy. Durban, South Africa. Proc. 15th Int. Cong. Electron Microscopy 2, 331–332.Google Scholar
  20. 20.
    Schräpler, V. R., Schmidt, T., Schultka, R., and Hepp, W-D. (1991) Farbstoffanalytische Untersuchungen zum polarisationsmikropischen Nachweis von Kollagen mit Solaminrot 4B (Teil II). Acta Histochem. 90, 75–85.PubMedGoogle Scholar
  21. 21.
    Milthorpe, B. K. (1994) Xenografts for tendon and ligament repair. Biomaterials 15, 745–752.PubMedCrossRefGoogle Scholar
  22. 22.
    Cox, G. C., Xu, P., Sheppard, C. J. R., and Ramshaw, J. (2003) Characterization of the second harmonic signal from collagen. Proceedings of SPIE 4963, 32–40.CrossRefGoogle Scholar
  23. 23.
    Zipfel, W. R., Williams, R. M., Christie, R., Nitikin, A. Y., Hyman, B. T., and Webb, W. W. (2003) Live tissue intrinsic emission microscopy using multiphoton excited native fluorescence and second harmonic generation. Proc. Natl. Acad. Sci. USA 100, 7075–7080.PubMedCrossRefGoogle Scholar
  24. 24.
    Manconi, F., Cox, G., Kable, E., Markham R., and Fraser, I. S. (2001) Computer-generated three-dimensional reconstruction of uterine histological parallel serial sections displaying microvascular and glandular structures in human endometrium. Micron 32, 449–453.PubMedCrossRefGoogle Scholar
  25. 25.
    Gorrell, M. D., Wang, X. M., Levy, M. T., et al. (2003) Intrahepatic expression of collagen and fibroblast activation protein (FAP) in hepatitis c virus infection. Adv. Exp. Med. Biol. 524, 235–243.PubMedCrossRefGoogle Scholar
  26. 26.
    Yariv, A. (1967) Quantum Electronics, Wiley, New York.Google Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Guy Cox
    • 1
  • Eleanor Kable
    • 1
  1. 1.Electron Microscope UnitUniversity of SydneySydneyAustralia

Personalised recommendations