OSA's Digital Library

Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 15, Iss. 14 — Jul. 9, 2007
  • pp: 8913–8924

Signal epidetection in third-harmonic generation microscopy of turbid media

Delphine Débarre, Nicolas Olivier, and Emmanuel Beaurepaire  »View Author Affiliations

Optics Express, Vol. 15, Issue 14, pp. 8913-8924 (2007)

View Full Text Article

Enhanced HTML    Acrobat PDF (556 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



Third-harmonic generation (THG) imaging of thick samples or large organisms requires TH light to be epicollected through the focusing objective. In this study we first estimate the amount of backward-to-forward TH radiation created by an isolated object as a function of size and spatial frequencies in the object. Theory and model experiments indicate that no significant signal can be epidetected from a (biological) dielectric structure embedded in a transparent medium. In contrast, backward emission is observed from metal nanoparticles where THG is partly a surface effect. We then address the case of an object embedded in a turbid medium. Experiments and Monte Carlo simulations show that epidetection is possible when the absorption mean free path of harmonic light in the medium exceeds its reduced scattering length, and that epicollection efficiency critically depends on the microscope field-of-view even at shallow depths, because backscattered light is essentially diffusive. These observations provide guidelines for optimizing epidetection in third-harmonic, second-harmonic, or CARS imaging of thick tissues.

© 2007 Optical Society of America

OCIS Codes
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(180.6900) Microscopy : Three-dimensional microscopy
(190.4160) Nonlinear optics : Multiharmonic generation
(290.7050) Scattering : Turbid media

ToC Category:
Medical Optics and Biotechnology

Original Manuscript: June 4, 2007
Revised Manuscript: June 16, 2007
Manuscript Accepted: June 22, 2007
Published: July 3, 2007

Virtual Issues
Vol. 2, Iss. 8 Virtual Journal for Biomedical Optics

Delphine Débarre, Nicolas Olivier, and Emmanuel Beaurepaire, "Signal epidetection in third-harmonic generation microscopy of turbid media," Opt. Express 15, 8913-8924 (2007)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. W. R. Zipfel, R. M. Williams and W. W. Webb, "Nonlinear magic:multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003). [CrossRef] [PubMed]
  2. K. Svoboda, W. Denk, D. Kleinfeld and D. W. Tank, "In vivo dendritic calcium dynamics in neocortical pyramidal neurons," Nature 385, 161-165 (1997). [CrossRef] [PubMed]
  3. F. Helmchen and W. Denk, "New developments in multiphoton microscopy", Curr. Opin. Neurobiol. 12, 593-601 (2002). [CrossRef] [PubMed]
  4. S. Charpak, J. Mertz, E. Beaurepaire, L. Moreaux and K. Delaney, "Odor-evoked calcium signals in dendrites of rat mitral cells," Proc. Nat. Acad. Sci. USA 98, 1230-1234 (2001). [CrossRef] [PubMed]
  5. M. D. Cahalan, I. Parker, S. H. Wei and M. J. Miller, "Real-time imaging of lymphocytes in vivo," Curr. Opin. Immunol. 15, 372-377 (2003). [CrossRef] [PubMed]
  6. S. Hugues, L. Fetler, L. Bonifaz, J. Helft, F. Amblard and S. Amigorena, "Distinct T cell dynamics in lymph nodes during the induction of tolerance and immunity," Nat. Immunol. 5, 1235-42 (2004). [CrossRef] [PubMed]
  7. W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, J. E. Segall and J. S. Condeelis, "Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling," Cancer Res. 62, 6278-88 (2002). [PubMed]
  8. J. M. Squirrell, D. L. Wokosin, J. G. White and B. D. Bavister, "Long-term two photon fluorescence imaging of mammalian embryos without compromising viability," Nat. Biotechnol. 17, 763-767 (1999). [CrossRef] [PubMed]
  9. W. Supatto, D. Débarre, B. Moulia, E. Brouzés, J.-L. Martin, E. Farge and E. Beaurepaire, "In vivo modulation of morphogenetic movements in Drosophila embryos with femtosecond laser pulses," Proc. Nat. Acad. Sci. USA 102, 1047-1052 (2005). [CrossRef] [PubMed]
  10. E. Beaurepaire and J. Mertz, "Epifluorescence collection in two-photon microscopy," Appl. Opt. 41, 5376-5382 (2002). [CrossRef] [PubMed]
  11. M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz and S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth," J. Neurosci. Methods 111, 29-37 (2001). [CrossRef] [PubMed]
  12. D. Vučinić, T. M. BartolJr. and T. J. Sejnowski, "Hybrid reflecting objectives for functional multiphoton microscopy in turbid media," Opt. Lett. 31, 2447-2449 (2006). [CrossRef] [PubMed]
  13. Y. Barad, H. Eisenberg, M. Horowitz and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-924 (1997). [CrossRef]
  14. M. Müller, J. Squier, K. R. Wilson and G. J. Brakenhoff, "3D-microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998). [CrossRef] [PubMed]
  15. D. Yelin and Y. Silberberg, "Laser scanning third-harmonic generation microscopy in biology," Opt. Express 5, 169-175 (1999). [CrossRef] [PubMed]
  16. D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima and Y. Silberberg, "Depth-resolved structural imaging by third-harmonic generation microscopy," J. Struct. Biol. 147, 3-11 (2004). [CrossRef] [PubMed]
  17. D. Débarre, W. Supatto, E. Farge, B. Moulia, M.-C. Schanne-Klein and E. Beaurepaire, "Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos," Opt. Lett. 29, 2881-2883 (2004). [CrossRef]
  18. D. Débarre, W. Supatto, A.-M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M.-C. Schanne-Klein and E. Beaurepaire, "Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy," Nat. Methods 3, 47-53 (2006). [CrossRef]
  19. C.-K. Sun, C.-C. Chen, S.-W. Chu, T.-H. Tsai, Y.-C. Chen and B.-L. Lin, "Multiharmonic generation biopsy of skin," Opt. Lett. 28, 2488-2490 (2003). [CrossRef] [PubMed]
  20. 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. Express 14, 6178-6187 (2006). [CrossRef] [PubMed]
  21. J.-X. Cheng and X. S. Xie, "Green's function formulation for third harmonic generation microscopy," J. Opt. Soc. Am. B 19, 1604-1610 (2002). [CrossRef]
  22. L. Moreaux, O. Sandre and J. Mertz, "Membrane imaging by second-harmonic generation microscopy," J. Opt. Soc. Am. B 17, 1685-1694 (2000). [CrossRef]
  23. J. Mertz and L. Moreaux, "Second-harmonic generation by focused excitation of inhomogeneously distributed scatterers," Opt. Commun. 196, 325-330 (2001). [CrossRef]
  24. R. M. Williams, W. R. Zipfel and W. W. Webb, "Interpreting second-harmonic images of collagen I fibrils," Biophys. J. 88, 1377-1386 (2005). [CrossRef]
  25. O. Nadiarnykh, R. Lacomb, P. J. Campagnola and W. A. Mohler, "Coherent and incoherent SHG in fibrillar cellulose matrices," Opt. Express 15, 3348-3360 (2007). [CrossRef] [PubMed]
  26. J.-X. Cheng, A. Volkmer and X. S. Xie, "Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy," J. Opt. Soc. Am. B 19, 1328-1347 (2002). [CrossRef]
  27. C. L. Evans, E. O. Potma, M. Puoris'haag, D. Côté, C. P. Lin and X. S. Xie, "Chemical imaging of tissue in vivo with video-rate coherent anti-STokes Raman scattering microscopy," Proc. Nat. Acad. Sci. USA 102, 16807-12 (2005). [CrossRef] [PubMed]
  28. R. W. Boyd, Nonlinear Optics, 2nd ed., (Academic Press, 2003).
  29. S.-P. Tai, T.-H. Tsai, W.-J. Lee, D.-B. Shieh, Y.-H. Liao, H.-Y. Huang, K. Y. J. Zhang, H.-L. Liu and C.-K. Sun, "Optical biopsy of fixed human skin with backward-collected optical harmonics signals," Opt. Express 13, 8231-8242 (2005). [CrossRef] [PubMed]
  30. D. Débarre, W. Supatto and E. Beaurepaire, "Structure sensitivity in third-harmonic generation microscopy," Opt. Lett. 30, 2134-2136 (2005). [CrossRef] [PubMed]
  31. B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanetic system," Proc. Royal Soc. London Ser. A 253, 358-379 (1959). [CrossRef]
  32. M. LippitzM. A. van Dijk.and M. Orrit, "Third-harmonic generation from single gold nanoparticules," Nano Lett. 5, 799-802 (2005). [CrossRef] [PubMed]
  33. S. V. Popruzhenko, D. F. Zaretsky and W. Becker, "Third-harmonic generation by small clusters in a dielectric medium," J. Phys. B. 39, 4933-4943 (2006). [CrossRef]
  34. J. J. Mock, M. Barbic, D. R. Smith, D. A. Schultz and S. Schultz, "Shape effects in plasmon resonance of individual silver nanoparticles," J. Chem. Phys. 116, 6755-6759 (2002). [CrossRef]
  35. M. H. Niemz, Laser-tissue interactions - Fundamentals and Applications - Third ed., (Springer, 2004).
  36. S. A. Prahl, M. Keijzer, S. L. Jacques and A. J. Welch, "A Monte Carlo model of light propagation in tissue," SPIE Institute Series 5, 102-111 (1989).
  37. C. G. Bohren and D. R. Huffman, Absorption and scattering of light by small particles, (Wiley, New York, 1983).
  38. V. Barzda, C. Greenhalgh, J. Aus-der-Au and S. Elmore, "Visualization of mitochondria in cardiomyocytes by simultaneous harmonic generation and fluorescence microscopy," Opt. Express 13, 8263-8276 (2006). [CrossRef]
  39. O. G. Clay, A. C. Millard, C. B. Schaffer, J. Aus-Der-Au, P. S. Tsai, J. A. Squier and D. Kleinfeld, "Spectroscopy of third harmonic generation: evidence for resonances in model compounds and ligated hemoglobin," J. Opt. Soc. Am. B 23, 932-950 (2006). [CrossRef]
  40. N. Djaker, D. Gachet, N. Sandeau, P.-F. Lenne and H. Rigneault, "Refractive effects in coherent anti-Stokes Raman scattering microscopy," Appl. Opt. 45, 7005-7011 (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.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited