OSA's Digital Library

Applied Optics

Applied Optics


  • Vol. 42, Iss. 25 — Sep. 1, 2003
  • pp: 5209–5219

Quantitative second-harmonic generation microscopy in collagen

Patrick Stoller, Peter M. Celliers, Karen M. Reiser, and Alexander M. Rubenchik  »View Author Affiliations

Applied Optics, Vol. 42, Issue 25, pp. 5209-5219 (2003)

View Full Text Article

Enhanced HTML    Acrobat PDF (1346 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



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

OCIS Codes
(170.0180) Medical optics and biotechnology : Microscopy
(170.5810) Medical optics and biotechnology : Scanning microscopy
(170.7160) Medical optics and biotechnology : Ultrafast technology
(180.5810) Microscopy : Scanning microscopy
(190.1900) Nonlinear optics : Diagnostic applications of nonlinear optics
(190.4710) Nonlinear optics : Optical nonlinearities in organic materials

Original Manuscript: September 10, 2002
Revised Manuscript: January 6, 2003
Published: September 1, 2003

Patrick Stoller, Peter M. Celliers, Karen M. Reiser, and Alexander M. Rubenchik, "Quantitative second-harmonic generation microscopy in collagen," Appl. Opt. 42, 5209-5219 (2003)

Sort:  Author  |  Year  |  Journal  |  Reset  


  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]
  3. 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 .
  4. Y. Guo, P. P. Ho, A. Tirksliunas, F. Lui, R. R. Alfano, “Optical harmonic generation from animal tissues by the use of picosecond and femtosecond laser pulses,” Appl. Opt. 35, 6810–6813 (1996). [CrossRef] [PubMed]
  5. 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]
  6. 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]
  7. 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]
  8. K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200, 83–104 (2000). [CrossRef] [PubMed]
  9. 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]
  10. 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]
  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]
  12. R. M. Williams, W. R. Zipfel, W. W. Webb, “Multiphoton microscopy in biological research,” Curr. Opin. Chem. Biol. 5, 603–608 (2001). [CrossRef] [PubMed]
  13. 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]
  14. S. Fine, W. P. Hansen, “Optical second harmonic generation in biological tissues,” Appl. Opt. 10, 2350–2353 (1971). [CrossRef] [PubMed]
  15. 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.
  16. S. Roth, I. Freund, “Optical second-harmonic scattering in rat-tail tendon,” Biopolymers 20, 1271–1290 (1981). [CrossRef] [PubMed]
  17. I. Freund, M. Deutsch, “Macroscopic polarity of connective tissue is due to discrete polar structures,” Biopolymers 25, 601–606 (1986). [CrossRef] [PubMed]
  18. 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]
  19. 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]
  20. L. Moreaux, O. Sandre, J. Mertz, “Membrane imaging by second-harmonic generation microscopy,” J. Opt. Soc. Am. B 17, 1685–1694 (2000). [CrossRef]
  21. 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]
  22. F. P. Bolin, L. E. Preuss, R. C. Taylor, R. J. Ference, “Refractive index of some mammalian tissues using a fiber optic cladding method,” Appl. Opt. 28, 2297–2303 (1989). [CrossRef] [PubMed]
  23. 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.
  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).
  27. 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]
  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.

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article

OSA is a member of CrossRef.

CrossCheck Deposited