OSA's Digital Library

Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 4, Iss. 11 — Nov. 1, 2013
  • pp: 2546–2554

Application of quantitative second-harmonic generation microscopy to dynamic conditions

Mohammad M. Kabir, V. V. G. Krishna Inavalli, Tung-Yuen Lau, and Kimani C. Toussaint, Jr.  »View Author Affiliations

Biomedical Optics Express, Vol. 4, Issue 11, pp. 2546-2554 (2013)

View Full Text Article

Enhanced HTML    Acrobat PDF (1610 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



We present a quantitative second-harmonic generation (SHG) imaging technique that quantifies the 2D spatial organization of collagen fiber samples under dynamic conditions, as an image is acquired. The technique is demonstrated for both a well-aligned tendon sample and a randomly aligned, sparsely distributed collagen scaffold sample. For a fixed signal-to-noise ratio, we confirm the applicability of this method for various window sizes (pixel areas) as well as with using a gridded overlay map that allows for correlations of fiber orientations within a given image. This work has direct impact to in vivo biological studies by incorporating simultaneous SHG image acquisition and analysis.

© 2013 Optical Society of America

OCIS Codes
(100.2960) Image processing : Image analysis
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:

Original Manuscript: July 2, 2013
Revised Manuscript: August 22, 2013
Manuscript Accepted: October 14, 2013
Published: October 21, 2013

Virtual Issues
Novel Techniques in Microscopy (2013) Biomedical Optics Express

Mohammad M. Kabir, V. V. G. Krishna Inavalli, Tung-Yuen Lau, and Kimani C. Toussaint, "Application of quantitative second-harmonic generation microscopy to dynamic conditions," Biomed. Opt. Express 4, 2546-2554 (2013)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. P. A. Franken, A. E. Hill, C. W. Peters, G. Weinreich, “Generation of Optical Harmonics,” Phys. Rev. Lett. 7(4), 118–119 (1961). [CrossRef]
  2. S. Tokutake, Y. Imanishi, M. Sisido, “Efficiency of Second Harmonic Generation from Amino Acids, Peptides, and Polypeptides Carrying Polarizable Aromatic Groups,” Mol. Cryst. Liq. Cryst. (Phila. Pa.) 170, 245–257 (1989).
  3. J. N. Gannaway, C. J. R. Sheppard, “Second-harmonic imaging in the scanning optical microscope,” Opt. Quantum Electron. 10(5), 435–439 (1978). [CrossRef]
  4. R. Hellwarth, P. Christensen, “Nonlinear optical microscopic examination of structure in polycrystalline ZnSe,” Opt. Commun. 12(3), 318–322 (1974). [CrossRef]
  5. I. Freund, M. Deutsch, “Second-harmonic microscopy of biological tissue,” Opt. Lett. 11(2), 94–96 (1986). [CrossRef] [PubMed]
  6. P. J. Campagnola, L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21(11), 1356–1360 (2003). [CrossRef] [PubMed]
  7. M. Strupler, A. M. Pena, M. Hernest, P. L. Tharaux, J. L. Martin, E. Beaurepaire, M. C. Schanne-Klein, “Second harmonic imaging and scoring of collagen in fibrotic tissues,” Opt. Express 15(7), 4054–4065 (2007). [CrossRef] [PubMed]
  8. F. Tiaho, G. Recher, D. Rouède, “Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy,” Opt. Express 15(19), 12286–12295 (2007). [CrossRef] [PubMed]
  9. R. LaComb, O. Nadiarnykh, P. J. Campagnola, “Quantitative Second Harmonic Generation Imaging of the Diseased State Osteogenesis Imperfecta: Experiment and Simulation,” Biophys. J. 94(11), 4504–4514 (2008). [CrossRef] [PubMed]
  10. X. Chen, O. Nadiarynkh, S. Plotnikov, P. J. Campagnola, “Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure,” Nat. Protoc. 7(4), 654–669 (2012). [CrossRef] [PubMed]
  11. T. Hompland, A. Erikson, M. Lindgren, T. Lindmo, C. de Lange Davies, “Second-harmonic generation in collagen as a potential cancer diagnostic parameter,” J. Biomed. Opt. 13(5), 054050 (2008). [CrossRef] [PubMed]
  12. P. G. Ellingsen, M. B. Lilledahl, L. M. S. Aas, C. L. Davies, M. Kildemo, “Quantitative characterization of articular cartilage using Mueller matrix imaging and multiphoton microscopy,” J. Biomed. Opt. 16(11), 116002 (2011). [CrossRef] [PubMed]
  13. M. B. Lilledahl, D. M. Pierce, T. Ricken, G. A. Holzapfel, C. L. Davies, “Structural analysis of articular cartilage using multiphoton microscopy: Input for biomechanical modeling,” IEEE Trans. Med. Imaging 30(9), 1635–1648 (2011). [CrossRef] [PubMed]
  14. M. B. Lilledahl, O. A. Haugen, C. de Lange Davies, L. O. Svaasand, “Characterization of vulnerable plaques by multiphoton microscopy,” J. Biomed. Opt. 12(4), 044005 (2007). [CrossRef] [PubMed]
  15. R. A. Rao, M. R. Mehta, K. C. Toussaint., “Fourier transform-second-harmonic generation imaging of biological tissues,” Opt. Express 17(17), 14534–14542 (2009). [CrossRef] [PubMed]
  16. R. A. R. Rao, M. R. Mehta, S. Leithem, K. C. Toussaint., “Quantitative analysis of forward and backward second-harmonic images of collagen fibers using Fourier transform second-harmonic-generation microscopy,” Opt. Lett. 34(24), 3779–3781 (2009). [CrossRef] [PubMed]
  17. M. Sivaguru, S. Durgam, R. Ambekar, D. Luedtke, G. Fried, A. Stewart, K. C. Toussaint., “Quantitative analysis of collagen fiber organization in injured tendons using Fourier transform-second harmonic generation imaging,” Opt. Express 18(24), 24983–24993 (2010). [CrossRef] [PubMed]
  18. R. Ambekar, M. Chittenden, I. Jasiuk, K. C. Toussaint., “Quantitative second-harmonic generation microscopy for imaging porcine cortical bone: Comparison to SEM and its potential to investigate age-related changes,” Bone 50(3), 643–650 (2012). [CrossRef] [PubMed]
  19. R. Ambekar, T. Y. Lau, M. Walsh, R. Bhargava, K. C. Toussaint., “Quantifying collagen structure in breast biopsies using second-harmonic generation imaging,” Biomed. Opt. Express 3(9), 2021–2035 (2012). [CrossRef] [PubMed]
  20. T. Y. Lau, R. Ambekar, K. C. Toussaint, “Quantification of collagen fiber organization using three-dimensional Fourier transform-second-harmonic generation imaging,” Opt. Express 20(19), 21821–21832 (2012). [CrossRef] [PubMed]
  21. T. Y. Lau, H. K. Sangha, E. K. Chien, B. L. McFarlin, A. J. Wagoner Johnson, K. C. Toussaint., “Application of Fourier transform-second-harmonic generation imaging to the rat cervix,” J. Microsc. 251(1), 77–83 (2013). [CrossRef] [PubMed]
  22. S. H. Huang, C. D. Hsiao, D. S. Lin, C. Y. Chow, C. J. Chang, I. Liau, “Imaging of zebrafish in Vivo with second-harmonic generation reveals shortened sarcomeres associated with myopathy induced by statin,” PLoS ONE 6(9), e24764 (2011). [CrossRef] [PubMed]
  23. B. E. Cohen, “Biological imaging: Beyond fluorescence,” Nature 467(7314), 407–408 (2010). [CrossRef] [PubMed]
  24. P. Pantazis, J. Maloney, D. Wu, S. E. Fraser, “Second harmonic generating (SHG) nanoprobes for in vivo imaging,” Proc. Natl. Acad. Sci. U.S.A. 107(33), 14535–14540 (2010). [CrossRef] [PubMed]
  25. C. B. Raub, V. Suresh, T. Krasieva, J. Lyubovitsky, J. D. Mih, A. J. Putnam, B. J. Tromberg, S. C. George, “Noninvasive Assessment of Collagen Gel Microstructure and Mechanics Using Multiphoton Microscopy,” Biophys. J. 92(6), 2212–2222 (2007). [CrossRef] [PubMed]
  26. G. Stockman and L. G. Shapiro, Computer Vision (Prentice Hall PTR, 2001).
  27. R. Gonzalez and R. Woods, Digital Image Processing (3rd Edition) (Prentice Hall, 2007).
  28. N. I. Fisher, Statistical Analysis of Circular Data (Cambridge University Press, Cambridge, 1995).
  29. A. S. S. R. Jammalamadaka, Topics in Circular Statistics (World Scientific, Singapore, 2001).
  30. D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, W. W. Webb, “Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7081–7086 (2003). [CrossRef] [PubMed]
  31. J. P. Ogilvie, D. Débarre, X. Solinas, J. L. Martin, E. Beaurepaire, M. Joffre, “Use of coherent control for selective two-photon fluorescence microscopy in live organisms,” Opt. Express 14(2), 759–766 (2006). [CrossRef] [PubMed]

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.


Fig. 1 Fig. 2 Fig. 3
Fig. 4 Fig. 5

Supplementary Material

» Media 1: MP4 (2197 KB)     
» Media 2: MP4 (1744 KB)     
» Media 3: MP4 (1651 KB)     
» Media 4: MP4 (2339 KB)     

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited