OSA's Digital Library

Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 5, Iss. 7 — Jul. 1, 2014
  • pp: 2231–2246

Molecular orientation sensitive second harmonic microscopy by radially and azimuthally polarized light

Tobias Ehmke, Tim Heiko Nitzsche, Andreas Knebl, and Alexander Heisterkamp  »View Author Affiliations

Biomedical Optics Express, Vol. 5, Issue 7, pp. 2231-2246 (2014)

View Full Text Article

Enhanced HTML    Acrobat PDF (4471 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



We demonstrate the possibility to switch the z-polarization component of the illumination in the vicinity of the focus of high-NA objective lenses by applying radially and azimuthally polarized incident light. The influence of the field distribution on nonlinear effects was first investigated by the means of simulations. These were performed for high-NA objective lenses commonly used in nonlinear microscopy. Special attention is paid to the influence of the polarization of the incoming field. For linearly, circularly and radially polarized light a considerable polarization component in z-direction is generated by high NA focusing. Azimuthal polarization is an exceptional case: even for strong focusing no z-component arises. Furthermore, the influence of the input polarization on the intensity contributing to the nonlinear signal generation was computed. No distinct difference between comparable input polarization states was found for chosen thresholds of nonlinear signal generation. Differences in signal generation for radially and azimuthally polarized vortex beams were experimentally evaluated in native collagen tissue (porcine cornea). The findings are in good agreement with the theoretical predictions and display the possibility to probe the molecular orientation along the optical axis of samples with known nonlinear properties. The combination of simulations regarding the nonlinear response of materials and experiments with different sample orientations and present or non present z-polarization could help to increase the understanding of nonlinear signal formation in yet unstudied materials.

© 2014 Optical Society of America

OCIS Codes
(000.3860) General : Mathematical methods in physics
(190.0190) Nonlinear optics : Nonlinear optics
(190.2620) Nonlinear optics : Harmonic generation and mixing
(260.5430) Physical optics : Polarization
(180.4315) Microscopy : Nonlinear microscopy
(190.4975) Nonlinear optics : Parametric processes

ToC Category:

Original Manuscript: March 28, 2014
Revised Manuscript: May 24, 2014
Manuscript Accepted: May 27, 2014
Published: June 12, 2014

Tobias Ehmke, Tim Heiko Nitzsche, Andreas Knebl, and Alexander Heisterkamp, "Molecular orientation sensitive second harmonic microscopy by radially and azimuthally polarized light," Biomed. Opt. Express 5, 2231-2246 (2014)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. R. M. Williams, W. R. Zipfel, and W. W. Webb, “Mulitphoton microscopy in biological research,” Chem. Biol.5, 603–608 (2001).
  2. W. Denk and K. Svoboda, “Photon upmanship: Why multiphoton imaging is more than a gimmick,” Neuron18, 351–357 (1997). [CrossRef] [PubMed]
  3. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nature Biotechnol.21, 1369–1377 (2003). [CrossRef]
  4. F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nature Methods2, 932–940 (2005). [CrossRef] [PubMed]
  5. N. Olivier, M. A. Luengo-Oroz, L. Duloquin, E. Faure, T. Savy, I. Veilleux, X. Solinas, D. Dbarre, P. Bourgine, A. Santos, N. Peyriras, and E. Beaurepaire, “Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy,” Science329, 967–971 (2010). [CrossRef] [PubMed]
  6. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248, 73–76 (1990). [CrossRef] [PubMed]
  7. N. Olivier, F. Aptel, K. Plamann, M.-C. Schanne-Klein, and E. Beaurepaire, “Harmonic microscopy of isotropic and anisotropic microstructure of the human cornea,” Opt. Express18, 5028–5040 (2010). [CrossRef] [PubMed]
  8. P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Ann. Rev. Biomed. Eng.2, 399–429 (2000). [CrossRef]
  9. L. Fu and M. Gu, “Fibre-optic nonlinear optical microscopy and endoscopy,” J. Microscopy226, 195–206 (2007). [CrossRef]
  10. R. W. Boyd, Nonlinear Optics, 3rd ed. (Elsevier Inc., 2008).
  11. A.-M. Pena, T. Boulesteix, T. Dartigalongue, and M.-C. Schanne-Klein, “Chiroptical effects in the second harmonic signal of collagens i and iv,” J. Am. Chem. Soc.127, 10314–10322 (2005). [CrossRef] [PubMed]
  12. 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, 1–15 (2011). [CrossRef]
  13. 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.115, 12759–12769 (2011). [CrossRef]
  14. R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett.91, 233901 (2003). [CrossRef] [PubMed]
  15. D. P. Biss and T. G. Brown, “Polarization-vortex-driven second-harmonic generation,” Opt. Lett.28, 923–925 (2003). [CrossRef] [PubMed]
  16. E. Y. S. Yew and C. J. R. Sheppard, “Second harmonic generation polarization microscopy with tightly focused linearly and radially polarized beams,” Opt. Commun.275, 453–457 (2007). [CrossRef]
  17. K. Yoshiki, M. Hashimoto, and T. Araki, “Second-harmonic-generation microscopy using excitation beam with controlled polarization pattern to determine three-dimensional molecular orientation,” Jpn. J. Appl. Phys.44, 1066–1068 (2005). [CrossRef]
  18. K. Yoshiki, K. Ryosuke, M. Hashimoto, N. Hashimoto, and T. Araki, “Second-harmonic-generation microscope using eight-segment polarization-mode converter to observe three-dimensional molecular orientation,” Opt. Lett.32, 1680–1682 (2007). [CrossRef] [PubMed]
  19. M. Leutenegger, R. Rao, R. A. Leitgeb, and T. Lasser, “Fast focus field calculations,” Opt. Express14, 11277–11291 (2006). [CrossRef] [PubMed]
  20. E. Wolf, ed., An Integral Representation of the Image Field, vol. 253 of A(Proceedings of the Royal Society of London - Mathematical and Physical Sciences, 1959).
  21. J.-C. Diels and W. Rudolph, Ultrashort Laser Pulse Phenomena: Fundamentals, Techniques and Applications on a Femtosecond Time Scale (Elsevier Inc., 2006).
  22. B. R. Masters, “Correlation of histology and linear and nonlinear microscopy of the living human cornea,” J. Biophoton.2, 127–139 (2009). [CrossRef]
  23. 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. Visual Sci.51, 2459–2465 (2010). [CrossRef]
  24. A. J. Quantock and R. D. Young, “Development of the corneal stroma, and the collagen-proteoglycan associations that help define its structure and function,” Developmental Dynamics237, 2607–2621 (2008). [CrossRef] [PubMed]
  25. M. D. Shoulders and R. T. Raines, “Collagen structure and stability,” Ann. Rev. Biochem.78, 929–958 (2009). [CrossRef] [PubMed]
  26. M. Beresna, “Polarization engineering with ultrafast laser writing in transparent media,” Ph.D. thesis, University of Southampton (2012).
  27. S. Richter, M. Heinrich, S. Doering, A. Tuennermann, S. Nolte, and U. Peschel, “Nanogratings in fused silica: Formation, control, and applications,” J. Laser Appl.24, 042008:1–8 (2012). [CrossRef]
  28. W. Radner, M. Zehetmayer, R. Aufreiter, and R. Mallinger, “Interlacing and cross-angle distribution of collagen lamellae in the human cornea,” Cornea17, 537–543 (1998). [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