OSA's Digital Library

Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 5, Iss. 4 — Apr. 1, 2014
  • pp: 1099–1113

Motion-artifact-robust, polarization-resolved second-harmonic-generation microscopy based on rapid polarization switching with electro-optic Pockells cell and its application to in vivo visualization of collagen fiber orientation in human facial skin

Yuji Tanaka, Eiji Hase, Shuichiro Fukushima, Yuki Ogura, Toyonobu Yamashita, Tetsuji Hirao, Tsutomu Araki, and Takeshi Yasui  »View Author Affiliations

Biomedical Optics Express, Vol. 5, Issue 4, pp. 1099-1113 (2014)

View Full Text Article

Enhanced HTML    Acrobat PDF (5321 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



Polarization-resolved second-harmonic-generation (PR-SHG) microscopy is a powerful tool for investigating collagen fiber orientation quantitatively with low invasiveness. However, the waiting time for the mechanical polarization rotation makes it too sensitive to motion artifacts and hence has hampered its use in various applications in vivo. In the work described in this article, we constructed a motion-artifact-robust, PR-SHG microscope based on rapid polarization switching at every pixel with an electro-optic Pockells cell (PC) in synchronization with step-wise raster scanning of the focus spot and alternate data acquisition of a vertical-polarization-resolved SHG signal and a horizontal-polarization-resolved one. The constructed PC-based PR-SHG microscope enabled us to visualize orientation mapping of dermal collagen fiber in human facial skin in vivo without the influence of motion artifacts. Furthermore, it implied the location and/or age dependence of the collagen fiber orientation in human facial skin. The robustness to motion artifacts in the collagen orientation measurement will expand the application scope of SHG microscopy in dermatology and collagen-related fields.

© 2014 Optical Society of America

OCIS Codes
(170.1870) Medical optics and biotechnology : Dermatology
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(190.4160) Nonlinear optics : Multiharmonic generation
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:

Original Manuscript: January 13, 2014
Revised Manuscript: February 16, 2014
Manuscript Accepted: March 2, 2014
Published: March 7, 2014

Yuji Tanaka, Eiji Hase, Shuichiro Fukushima, Yuki Ogura, Toyonobu Yamashita, Tetsuji Hirao, Tsutomu Araki, and Takeshi Yasui, "Motion-artifact-robust, polarization-resolved second-harmonic-generation microscopy based on rapid polarization switching with electro-optic Pockells cell and its application to in vivo visualization of collagen fiber orientation in human facial skin," Biomed. Opt. Express 5, 1099-1113 (2014)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. A. Yariv, Introduction to Optical Electronics, (Holt McDougal, 1977).
  2. S. Fine and W. P. Hansen, “Optical second harmonic generation in biological systems,” Appl. Opt.10(10), 2350–2353 (1971). [CrossRef] [PubMed]
  3. W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A.100(12), 7075–7080 (2003). [CrossRef] [PubMed]
  4. 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), 493–508 (2002). [CrossRef] [PubMed]
  5. K. König and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt.8(3), 432–439 (2003). [CrossRef] [PubMed]
  6. M. J. Koehler, K. König, P. Elsner, R. Bückle, and M. Kaatz, “In vivo assessment of human skin aging by multiphoton laser scanning tomography,” Opt. Lett.31(19), 2879–2881 (2006). [CrossRef] [PubMed]
  7. J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “In vivo nonlinear spectral imaging in mouse skin,” Opt. Express14(10), 4395–4402 (2006). [CrossRef] [PubMed]
  8. T. Yasui, Y. Takahashi, M. Ito, S. Fukushima, and T. Araki, “Ex vivo and in vivo second-harmonic-generation imaging of dermal collagen fiber in skin: comparison of imaging characteristics between mode-locked Cr:Forsterite and Ti:Sapphire lasers,” Appl. Opt.48(10), D88–D95 (2009). [CrossRef] [PubMed]
  9. S.-Y. Chen, H.-Y. Wu, and C.-K. Sun, “In vivo harmonic generation biopsy of human skin,” J. Biomed. Opt.14(6), 060505 (2009). [CrossRef] [PubMed]
  10. S.-H. Chia, C.-H. Yu, C.-H. Lin, N.-C. Cheng, T.-M. Liu, M.-C. Chan, I.-H. Chen, and C.-K. Sun, “Miniaturized video-rate epi-third-harmonic-generation fiber-microscope,” Opt. Express18(16), 17382–17391 (2010). [CrossRef] [PubMed]
  11. T. Yasui, M. Yonetsu, R. Tanaka, Y. Tanaka, S. Fukushima, T. Yamashita, Y. Ogura, T. Hirao, H. Murota, and T. Araki, “In vivo observation of age-related structural changes of dermal collagen in human facial skin using collagen-sensitive second harmonic generation microscope equipped with 1250-nm mode-locked Cr:Forsterite laser,” J. Biomed. Opt.18(3), 031108 (2013). [CrossRef] [PubMed]
  12. R. Tanaka, S. Fukushima, K. Sasaki, Y. Tanaka, H. Murota, T. Matsumoto, T. Araki, and T. Yasui, “In vivo visualization of dermal collagen fiber in skin burn by collagen-sensitive second-harmonic-generation microscopy,” J. Biomed. Opt.18(6), 061231 (2013). [CrossRef] [PubMed]
  13. P. Stoller, B.-M. Kim, A. M. Rubenchik, K. M. Reiser, and L. B. Da Silva, “Polarization-dependent optical second-harmonic imaging of a rat-tail tendon,” J. Biomed. Opt.7(2), 205–214 (2002). [CrossRef] [PubMed]
  14. 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]
  15. T. Yasui, Y. Tohno, and T. Araki, “Characterization of collagen orientation in human dermis by two-dimensional second-harmonic-generation polarimetry,” J. Biomed. Opt.9(2), 259–264 (2004). [CrossRef] [PubMed]
  16. T. Yasui, Y. Tohno, and T. Araki, “Determination of collagen fiber orientation in human tissue by use of polarization measurement of molecular second-harmonic-generation light,” Appl. Opt.43(14), 2861–2867 (2004). [CrossRef] [PubMed]
  17. T. Yasui, K. Sasaki, Y. Tohno, and T. Araki, “Tomographic imaging of collagen fiber orientation in human tissue using depth-resolved polarimetry of second-harmonic-generation light,” Opt. Quantum Electron.37(13-15), 1397–1408 (2005). [CrossRef]
  18. J. C. Mansfield, C. P. Winlove, J. Moger, and S. J. Matcher, “Collagen fiber arrangement in normal and diseased cartilage studied by polarization sensitive nonlinear microscopy,” J. Biomed. Opt.13(4), 044020 (2008). [CrossRef] [PubMed]
  19. T. Yasui, Y. Takahashi, S. Fukushima, Y. Ogura, T. Yamashita, T. Kuwahara, T. Hirao, and T. Araki, “Observation of dermal collagen fiber in wrinkled skin using polarization-resolved second-harmonic-generation microscopy,” Opt. Express17(2), 912–923 (2009). [CrossRef] [PubMed]
  20. G. Latour, I. Gusachenko, L. Kowalczuk, I. Lamarre, and M.-C. Schanne-Klein, “In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy,” Biomed. Opt. Express3(1), 1–15 (2012). [CrossRef] [PubMed]
  21. I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources,” Opt. Quantum Electron.34(12), 1251–1266 (2002). [CrossRef]
  22. S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, H.-J. Tsai, and C.-K. Sun, “In vivo developmental biology study using noninvasive multi-harmonic generation microscopy,” Opt. Express11(23), 3093–3099 (2003). [CrossRef] [PubMed]
  23. S.-P. Tai, W.-J. Lee, D.-B. Shieh, P.-C. Wu, H.-Y. Huang, C.-H. Yu, and C.-K. Sun, “In vivo optical biopsy of hamster oral cavity with epi-third-harmonic-generation microscopy,” Opt. Express14(13), 6178–6187 (2006). [CrossRef] [PubMed]
  24. R. A. 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]
  25. 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]
  26. A. Ghazaryan, H. F. Tsai, G. Hayrapetyan, W.-L. Chen, Y.-F. Chen, M. Y. Jeong, C.-S. Kim, S.-J. Chen, and C.-Y. Dong, “Analysis of collagen fiber domain organization by Fourier second harmonic generation microscopy,” J. Biomed. Opt.18(3), 031105 (2013). [CrossRef] [PubMed]
  27. S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys. J.90(2), 693–703 (2006). [CrossRef] [PubMed]
  28. P. J. Campagnola and C.-Y. Dong, “Second harmonic generation microscopy: principles and applications to disease diagnosis,” Laser Photon. Rev.5(1), 13–26 (2011). [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.

Supplementary Material

» Media 1: MOV (937 KB)     
» Media 2: MOV (789 KB)     

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited