OSA's Digital Library

Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 4, Iss. 10 — Oct. 1, 2013
  • pp: 1991–2002

Precise, motion-free polarization control in Second Harmonic Generation microscopy using a liquid crystal modulator in the infinity space

Chi-Hsiang Lien, Karissa Tilbury, Shean-Jen Chen, and Paul J. Campagnola  »View Author Affiliations

Biomedical Optics Express, Vol. 4, Issue 10, pp. 1991-2002 (2013)

View Full Text Article

Enhanced HTML    Acrobat PDF (2975 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



Second Harmonic Generation (SHG) microscopy coupled with polarization analysis has great potential for use in tissue characterization, as molecular and supramolecular structural details can be extracted. Such measurements are difficult to perform quickly and accurately. Here we present a new method that uses a liquid crystal modulator (LCM) located in the infinity space of a SHG laser scanning microscope that allows the generation of any desired linear or circular polarization state. As the device contains no moving parts, polarization can be rotated accurately and faster than by manual or motorized control. The performance in terms of polarization purity was validated using Stokes vector polarimetry, and found to have minimal residual polarization ellipticity. SHG polarization imaging characteristics were validated against well-characterized specimens having cylindrical and/or linear symmetries. The LCM has a small footprint and can be implemented easily in any standard microscope and is cost effective relative to other technologies.

© 2013 OSA

OCIS Codes
(180.6900) Microscopy : Three-dimensional microscopy
(190.2620) Nonlinear optics : Harmonic generation and mixing
(180.4315) Microscopy : Nonlinear microscopy
(110.5405) Imaging systems : Polarimetric imaging
(170.6935) Medical optics and biotechnology : Tissue characterization

ToC Category:

Original Manuscript: July 5, 2013
Revised Manuscript: August 22, 2013
Manuscript Accepted: August 23, 2013
Published: September 5, 2013

Virtual Issues
Optical Molecular Probes, Imaging, and Drug Delivery (2013) Biomedical Optics Express

Chi-Hsiang Lien, Karissa Tilbury, Shean-Jen Chen, and Paul J. Campagnola, "Precise, motion-free polarization control in Second Harmonic Generation microscopy using a liquid crystal modulator in the infinity space," Biomed. Opt. Express 4, 1991-2002 (2013)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. 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]
  2. W. Lo, S. W. Teng, H. Y. Tan, K. H. Kim, H. C. Chen, H. S. Lee, Y. F. Chen, P. T. C. So, and C. Y. Dong, “Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging,” Microsc. Res. Tech.69(12), 973–975 (2006). [CrossRef] [PubMed]
  3. P. P. Provenzano, D. R. Inman, K. W. Eliceiri, J. G. Knittel, L. Yan, C. T. Rueden, J. G. White, and P. J. Keely, “Collagen density promotes mammary tumor initiation and progression,” BMC Med.6(1), 11 (2008). [CrossRef] [PubMed]
  4. S. Y. Chen, S. U. Chen, H. Y. Wu, W. J. Lee, Y. H. Liao, and C. K. Sun, “In Vivo virtual biopsy of human skin by using noninvasive higher harmonic generation microscopy,” IEEE J. Sel. Top. Quantum Electron.16(3), 478–492 (2010). [CrossRef]
  5. E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med.9(6), 796–801 (2003). [CrossRef] [PubMed]
  6. R. Cicchi, S. Sestini, V. De Giorgi, P. Carli, D. Massi, and F. S. Pavone, “Basal cell carcinoma imaging and characterization by multiple nonlinear microscopy techniques,” Biophys. J.157a–157a (2007).
  7. M. Strupler, A. M. Pena, M. Hernest, P. L. Tharaux, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Second harmonic imaging and scoring of collagen in fibrotic tissues,” Opt. Express15(7), 4054–4065 (2007). [CrossRef] [PubMed]
  8. C. P. Pfeffer, B. R. Olsen, and F. Légaré, “Second harmonic generation imaging of fascia within thick tissue block,” Opt. Express15(12), 7296–7302 (2007). [CrossRef] [PubMed]
  9. K. G. Brockbank, W. R. MacLellan, J. Xie, S. F. Hamm-Alvarez, Z. Z. Chen, and K. Schenke-Layland, “Quantitative second harmonic generation imaging of cartilage damage,” Cell Tissue Bank.9(4), 299–307 (2008). [CrossRef] [PubMed]
  10. G. P. Kwon, J. L. Schroeder, M. J. Amar, A. T. Remaley, and R. S. Balaban, “Contribution of macromolecular structure to the retention of low-density lipoprotein at arterial branch points,” Circulation117(22), 2919–2927 (2008). [CrossRef] [PubMed]
  11. S. V. Plotnikov, A. M. Kenny, S. J. Walsh, B. Zubrowski, C. Joseph, V. L. Scranton, G. A. Kuchel, D. Dauser, M. Xu, C. C. Pilbeam, D. J. Adams, R. P. Dougherty, P. J. Campagnola, and W. A. Mohler, “Measurement of muscle disease by quantitative second-harmonic generation imaging,” J. Biomed. Opt.13(4), 044018 (2008). [CrossRef] [PubMed]
  12. V. Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, L. Fusi, M. Linari, G. Piazzesi, V. Lombardi, and F. S. Pavone, “Probing myosin structural conformation in vivo by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A.107(17), 7763–7768 (2010). [CrossRef] [PubMed]
  13. P. Campagnola, “Second harmonic generation imaging microscopy: applications to diseases diagnostics,” Anal. Chem.83(9), 3224–3231 (2011). [CrossRef] [PubMed]
  14. 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]
  15. A. M. Pena, A. Fabre, D. Débarre, J. Marchal-Somme, B. Crestani, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy,” Microsc. Res. Tech.70(2), 162–170 (2007). [CrossRef] [PubMed]
  16. W. Sun, S. Chang, D. C. Tai, N. Tan, G. Xiao, H. Tang, and H. Yu, “Nonlinear optical microscopy: use of second harmonic generation and two-photon microscopy for automated quantitative liver fibrosis studies,” J. Biomed. Opt.13(6), 064010 (2008). [CrossRef] [PubMed]
  17. M. W. Conklin, J. C. Eickhoff, K. M. Riching, C. A. Pehlke, K. W. Eliceiri, P. P. Provenzano, A. Friedl, and P. J. Keely, “Aligned collagen is a prognostic signature for survival in human breast carcinoma,” Am. J. Pathol.178(3), 1221–1232 (2011). [CrossRef] [PubMed]
  18. 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]
  19. 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]
  20. A. E. Tuer, S. Krouglov, N. Prent, R. Cisek, D. Sandkuijl, K. Yasufuku, B. C. Wilson, and V. Barzda, “Nonlinear optical properties of type I collagen fibers studied by polarization dependent second harmonic generation microscopy,” J. Phys. Chem. B115(44), 12759–12769 (2011). [CrossRef] [PubMed]
  21. I. Gusachenko, G. Latour, and M. C. Schanne-Klein, “Polarization-resolved Second Harmonic microscopy in anisotropic thick tissues,” Opt. Express18(18), 19339–19352 (2010). [CrossRef] [PubMed]
  22. I. Gusachenko, V. Tran, Y. G. Houssen, J. M. Allain, and M. C. Schanne-Klein, “Polarization-resolved second-harmonic generation in tendon upon mechanical stretching,” Biophys. J.102(9), 2220–2229 (2012). [CrossRef] [PubMed]
  23. D. Rouède, J. J. Bellanger, E. Schaub, G. Recher, and F. Tiaho, “Theoretical and experimental SHG angular intensity patterns from healthy and proteolysed muscles,” Biophys. J.104(9), 1959–1968 (2013). [CrossRef] [PubMed]
  24. P. J. Su, W. L. Chen, Y. F. Chen, and C. Y. Dong, “Determination of collagen nanostructure from second-order susceptibility tensor analysis,” Biophys. J.100(8), 2053–2062 (2011). [CrossRef] [PubMed]
  25. S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, “Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy,” Biophys. J.86(6), 3914–3922 (2004). [CrossRef] [PubMed]
  26. S. Brasselet, “Polarization resolved nonlinear microscopy: application to structural molecular and biological imaging,” Adv. Opt. Photon.3(3), 205–271 (2011). [CrossRef]
  27. C. K. Chou, W. L. Chen, P. T. Fwu, S. J. Lin, H. S. Lee, and C. Y. Dong, “Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation,” J. Biomed. Opt.13(1), 014005 (2008). [CrossRef] [PubMed]
  28. P. Schön, F. Munhoz, A. Gasecka, S. Brustlein, and S. Brasselet, “Polarization distortion effects in polarimetric two-photon microscopy,” Opt. Express16(25), 20891–20901 (2008). [CrossRef] [PubMed]
  29. X. Chen, O. Nadiarynkh, S. Plotnikov, and P. J. Campagnola, “Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure,” Nat. Protoc.7(4), 654–669 (2012). [CrossRef] [PubMed]
  30. F. Tiaho, G. Recher, and D. Rouède, “Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy,” Opt. Express15(19), 12286–12295 (2007). [CrossRef] [PubMed]
  31. O. Nadiarnykh and P. J. Campagnola, “Retention of polarization signatures in SHG microscopy of scattering tissues through optical clearing,” Opt. Express17(7), 5794–5806 (2009). [CrossRef] [PubMed]
  32. O. Sandre, L. Moreaux, and F. Brochard-Wyart, “Dynamics of transient pores in stretched vesicles,” Proc. Natl. Acad. Sci. U.S.A.96(19), 10591–10596 (1999). [CrossRef] [PubMed]
  33. X. Y. Chen, C. Raggio, and P. J. Campagnola, “Second-harmonic generation circular dichroism studies of osteogenesis imperfecta,” Opt. Lett.37(18), 3837–3839 (2012). [CrossRef] [PubMed]
  34. A. C. Millard, L. Jin, M. D. Wei, J. P. Wuskell, A. Lewis, and L. M. Loew, “Sensitivity of second harmonic generation from styryl dyes to transmembrane potential,” Biophys. J.86(2), 1169–1176 (2004). [CrossRef] [PubMed]
  35. L. Moreaux, O. Sandre, S. Charpak, M. Blanchard-Desce, and J. Mertz, “Coherent scattering in multi-harmonic light microscopy,” Biophys. J.80(3), 1568–1574 (2001). [CrossRef] [PubMed]
  36. D. Axelrod, “Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization,” Biophys. J.26(3), 557–573 (1979). [CrossRef] [PubMed]
  37. R. Oldenbourg and G. Mei, “New polarized light microscope with precision universal compensator,” J. Microsc.180(2), 140–147 (1995). [CrossRef] [PubMed]
  38. X. Wang and L. V. Wang, “Propagation of polarized light in birefringent turbid media: a Monte Carlo study,” J. Biomed. Opt.7(3), 279–290 (2002). [CrossRef] [PubMed]
  39. J. Duboisset, D. Ait-Belkacem, M. Roche, H. Rigneault, and S. Brasselet, “Generic model of the molecular orientational distribution probed by polarization-resolved second-harmonic generation,” Phys. Rev. A85(4), 043829 (2012). [CrossRef]
  40. O. Nadiarnykh, S. Plotnikov, W. A. Mohler, I. Kalajzic, D. Redford-Badwal, and P. J. Campagnola, “Second harmonic generation imaging microscopy studies of Osteogenesis Imperfecta,” J. Biomed. Opt.12(5), 051805 (2007). [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.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited