OSA's Digital Library

Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 4, Iss. 7 — Jul. 1, 2013
  • pp: 1006–1013

Analysis of spatial lamellar distribution from adaptive-optics second harmonic generation corneal images

Juan M. Bueno, Raquel Palacios, Mary K. Chessey, and Harilaos Ginis  »View Author Affiliations

Biomedical Optics Express, Vol. 4, Issue 7, pp. 1006-1013 (2013)

View Full Text Article

Enhanced HTML    Acrobat PDF (1238 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



The spatial organization of stromal collagen of ex-vivo corneas has been quantified in adaptive-optics second harmonic generation (SHG) images by means of an optimized Fourier transform (FT) based analysis. At a particular depth location, adjacent lamellae often present similar orientations and run parallel to the corneal surface. However this pattern might be combined with interweaved collagen bundles leading to crosshatched structures with different orientations. The procedure here reported provides us with both principal and crosshatched angles. This is also able to automatically distinguish a random distribution from a cross-shaped one, since it uses the ratio of the axes lengths of the best-fitted ellipse of the FT data as an auxiliary parameter. The technique has successfully been applied to SHG images of healthy corneas (both stroma and Bowman’s layer) of different species and to corneas undergoing cross-linking treatment.

© 2013 OSA

OCIS Codes
(110.2960) Imaging systems : Image analysis
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(170.4470) Medical optics and biotechnology : Ophthalmology
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:
Image Processing

Original Manuscript: March 12, 2013
Revised Manuscript: April 24, 2013
Manuscript Accepted: April 24, 2013
Published: June 4, 2013

Juan M. Bueno, Raquel Palacios, Mary K. Chessey, and Harilaos Ginis, "Analysis of spatial lamellar distribution from adaptive-optics second harmonic generation corneal images," Biomed. Opt. Express 4, 1006-1013 (2013)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. A. T. Yeh, N. Nassif, A. Zoumi, and B. J. Tromberg, “Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence,” Opt. Lett.27(23), 2082–2084 (2002). [CrossRef] [PubMed]
  2. Y. Komai and T. Ushiki, “The three-dimensional organization of collagen fibrils in the human cornea and sclera,” Invest. Ophthalmol. Vis. Sci.32(8), 2244–2258 (1991). [PubMed]
  3. M. Han, G. Giese, and J. Bille, “Second harmonic generation imaging of collagen fibrils in cornea and sclera,” Opt. Express13(15), 5791–5797 (2005). [CrossRef] [PubMed]
  4. N. Morishige, A. J. Wahlert, M. C. Kenney, D. J. Brown, K. Kawamoto, T. Chikama, T. Nishida, and J. V. Jester, “Second-harmonic imaging microscopy of normal human and keratoconus cornea,” Invest. Ophthalmol. Vis. Sci.48(3), 1087–1094 (2007). [CrossRef] [PubMed]
  5. F. Aptel, N. Olivier, A. Deniset-Besseau, J.-M. Legeais, K. Plamann, M.-C. Schanne-Klein, and E. Beaurepaire, “Multimodal nonlinear imaging of the human cornea,” Invest. Ophthalmol. Vis. Sci.51(5), 2459–2465 (2010). [CrossRef] [PubMed]
  6. J. M. Bueno, E. J. Gualda, and P. Artal, “Analysis of corneal stroma organization with wavefront optimized nonlinear microscopy,” Cornea30(6), 692–701 (2011). [CrossRef] [PubMed]
  7. H. Y. Tan, Y. Sun, W. Lo, S. J. Lin, C. H. Hsiao, Y. F. Chen, S. C. Huang, W. C. Lin, S. H. Jee, H. S. Yu, and C. Y. Dong, “Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo,” Invest. Ophthalmol. Vis. Sci.47(12), 5251–5259 (2006). [CrossRef] [PubMed]
  8. K. Plamann, F. Aptel, C. L. Arnold, A. Courjaud, C. Crotti, F. Deloison, F. Druon, P. Georges, M. Hanna, J.-M. Legeais, F. Morin, É. Mottay, V. Nuzzo, D. A. Peyrot, and M. Savoldelli, “Ultrashort pulse laser surgery of the cornea and the sclera,” J. Opt.12(8), 084002 (2010). [CrossRef]
  9. E. J. Gualda, J. R. Vázquez de Aldana, M. C. Martínez-García, P. Moreno, J. Hernández-Toro, L. Roso, P. Artal, and J. M. Bueno, “Femtosecond infrared intrastromal ablation and backscattering-mode adaptive-optics multiphoton microscopy in chicken corneas,” Biomed. Opt. Express2(11), 2950–2960 (2011). [CrossRef] [PubMed]
  10. P. Steven, M. Hovakimyan, R. F. Guthoff, G. Hüttmann, and O. Stachs, “Imaging corneal crosslinking by autofluorescence 2-photon microscopy, second harmonic generation, and fluorescence lifetime measurements,” J. Cataract Refract. Surg.36(12), 2150–2159 (2010). [CrossRef] [PubMed]
  11. J. M. Bueno, E. J. Gualda, A. Giakoumaki, P. Pérez-Merino, S. Marcos, and P. Artal, “Multiphoton microscopy of ex vivo corneas after collagen cross-linking,” Invest. Ophthalmol. Vis. Sci.52(8), 5325–5331 (2011). [CrossRef] [PubMed]
  12. P. Matteini, F. Ratto, F. Rossi, R. Cicchi, C. Stringari, D. Kapsokalyvas, F. S. Pavone, and R. Pini, “Photothermally-induced disordered patterns of corneal collagen revealed by SHG imaging,” Opt. Express17(6), 4868–4878 (2009). [CrossRef] [PubMed]
  13. W. Lo, Y.-L. Chang, J.-S. Liu, C.-M. Hseuh, V. Hovhannisyan, S.-J. Chen, H.-Y. Tan, and C.-Y. Dong, “Multimodal, multiphoton microscopy and image correlation analysis for characterizing corneal thermal damage,” J. Biomed. Opt.14(5), 054003 (2009). [CrossRef] [PubMed]
  14. R. A. R. Rao, M. R. Mehta, and K. C. Toussaint., “Fourier transform-second-harmonic generation imaging of biological tissues,” Opt. Express17(17), 14534–14542 (2009). [CrossRef] [PubMed]
  15. W. Lo, W. L. Chen, C. M. Hsueh, A. A. Ghazaryan, S. J. Chen, D. H. K. Ma, C. Y. Dong, and H. Y. Tan, “Fast Fourier transform-based analysis of second-harmonic generation image in keratoconic cornea,” Invest. Ophthalmol. Vis. Sci.53(7), 3501–3507 (2012). [CrossRef] [PubMed]
  16. Y. Mega, M. Robitaille, R. Zareian, J. McLean, J. Ruberti, and C. DiMarzio, “Quantification of lamellar orientation in corneal collagen using second harmonic generation images,” Opt. Lett.37(16), 3312–3314 (2012). [CrossRef] [PubMed]
  17. N. Morishige, Y. Takagi, T. Chikama, A. Takahara, and T. Nishida, “Three-dimensional analysis of collagen lamellae in the anterior stroma of the human cornea visualized by second harmonic generation imaging microscopy,” Invest. Ophthalmol. Vis. Sci.52(2), 911–915 (2011). [CrossRef] [PubMed]
  18. M. Winkler, D. Chai, S. Kriling, C. J. Nien, D. J. Brown, B. Jester, T. Juhasz, and J. V. Jester, “Nonlinear optical macroscopic assessment of 3-D corneal collagen organization and axial biomechanics,” Invest. Ophthalmol. Vis. Sci.52(12), 8818–8827 (2011). [CrossRef] [PubMed]
  19. J. M. Bueno, E. J. Gualda, and P. Artal, “Adaptive optics multiphoton microscopy to study ex vivo ocular tissues,” J. Biomed. Opt.15(6), 066004 (2010). [CrossRef] [PubMed]
  20. G. Wollensak, “Crosslinking treatment of progressive keratoconus: new hope,” Curr. Opin. Ophthalmol.17(4), 356–360 (2006). [CrossRef] [PubMed]
  21. F. Rossi, P. Matteini, I. Bruno, P. Nesi, and R. Pini, “Monitoring thermally-induced phase transitions in porcine cornea with the use of fluorescence micro-imaging analysis,” Opt. Express15(18), 11178–11184 (2007). [CrossRef] [PubMed]
  22. K. Lu, J. Chen, S. Zhuo, L. Zheng, X. Jiang, X. Zhu, and J. Zhao, “Multiphoton laser scanning microscopy of localized scleroderma,” Skin Res. Technol.15(4), 489–495 (2009). [CrossRef] [PubMed]
  23. R. Cicchi, D. Kapsokalyvas, V. De Giorgi, V. Maio, A. Van Wiechen, D. Massi, T. Lotti, and F. S. Pavone, “Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy,” J. Biophotonics3(1-2), 34–43 (2010). [CrossRef] [PubMed]
  24. R. A. R. Rao, M. R. Mehta, S. Leithem, and 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]
  25. D. T. Fung, J. B. Sereysky, J. Basta-Pljakic, D. M. Laudier, R. Huq, K. J. Jepsen, M. B. Schaffler, and E. L. Flatow, “Second harmonic generation imaging and Fourier transform spectral analysis reveal damage in fatigue-loaded tendons,” Ann. Biomed. Eng.38(5), 1741–1751 (2010). [CrossRef] [PubMed]
  26. M. Sivaguru, S. Durgam, R. Ambekar, D. Luedtke, G. Fried, A. Stewart, and K. C. Toussaint., “Quantitative analysis of collagen fiber organization in injured tendons using Fourier transform-second harmonic generation imaging,” Opt. Express18(24), 24983–24993 (2010). [CrossRef] [PubMed]
  27. J. K. Pijanka, B. Coudrillier, K. Ziegler, T. Sorensen, K. M. Meek, T. D. Nguyen, H. A. Quigley, and C. Boote, “Quantitative mapping of collagen fiber orientation in non-glaucoma and glaucoma posterior human sclerae,” Invest. Ophthalmol. Vis. Sci.53(9), 5258–5270 (2012). [CrossRef] [PubMed]
  28. R. Ambekar, T.-Y. Lau, M. Walsh, R. Bhargava, and K. C. Toussaint., “Quantifying collagen structure in breast biopsies using second-harmonic generation imaging,” Biomed. Opt. Express3(9), 2021–2035 (2012). [CrossRef] [PubMed]
  29. C.-M. Hsueh, W. Lo, W.-L. Chen, V. A. Hovhannisyan, G.-Y. Liu, S.-S. Wang, H.-Y. Tan, and C.-Y. Dong, “Structural characterization of edematous corneas by forward and backward second harmonic generation imaging,” Biophys. J.97(4), 1198–1205 (2009). [CrossRef] [PubMed]
  30. Q. Wu, B. E. Applegate, and A. T. Yeh, “Cornea microstructure and mechanical responses measured with nonlinear optical and optical coherence microscopy using sub-10-fs pulses,” Biomed. Opt. Express2(5), 1135–1146 (2011). [CrossRef] [PubMed]
  31. J. M. Bueno, E. J. Gualda, and P. Artal, “Non-linear 3D microscopy of ex vivo corneas,” Proc. SPIE7569, 756937, 756937-4 (2010). [CrossRef]

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

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited