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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 21, Iss. 5 — Mar. 11, 2013
  • pp: 5998–6008

Two-photon excitation selective plane illumination microscopy (2PE-SPIM) of highly scattering samples: characterization and application

Zeno Lavagnino, Francesca Cella Zanacchi, Emiliano Ronzitti, and Alberto Diaspro  »View Author Affiliations

Optics Express, Vol. 21, Issue 5, pp. 5998-6008 (2013)

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In this work we report the advantages provided by two photon excitation (2PE) implemented in a selective plane illumination microscopy (SPIM) when imaging thick scattering samples. In particular, a detailed analysis of the effects induced on the real light sheet excitation intensity distribution is performed. The comparison between single-photon and two-photon excitation profiles shows the reduction of the scattering effects and sample-induced aberrations provided by 2PE-SPIM. Furthermore, uniformity of the excitation distribution and the consequent improved image contrast is shown when imaging scattering phantom samples in depth by 2PE-SPIM. These results show the advantages of 2PE-SPIM and suggest how this combination can further enhance the SPIM performance. Phantom samples have been designed with optical properties compatible with biological applications of interest.

© 2013 OSA

OCIS Codes
(180.0180) Microscopy : Microscopy
(110.0113) Imaging systems : Imaging through turbid media

ToC Category:

Original Manuscript: November 29, 2012
Revised Manuscript: January 26, 2013
Manuscript Accepted: February 16, 2013
Published: March 4, 2013

Virtual Issues
Vol. 8, Iss. 4 Virtual Journal for Biomedical Optics

Zeno Lavagnino, Francesca Cella Zanacchi, Emiliano Ronzitti, and Alberto Diaspro, "Two-photon excitation selective plane illumination microscopy (2PE-SPIM) of highly scattering samples: characterization and application," Opt. Express 21, 5998-6008 (2013)

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  1. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004). [CrossRef] [PubMed]
  2. P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods4(4), 311–313 (2007). [PubMed]
  3. P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010). [CrossRef] [PubMed]
  4. M. Friedrich, Q. Gan, V. Ermolayev, and G. S. Harms, “STED-SPIM: Stimulated emission depletion improves sheet illumination microscopy resolution,” Biophys. J.100(8), L43–L45 (2011). [CrossRef] [PubMed]
  5. F. Cella Zanacchi, Z. Lavagnino, M. Perrone Donnorso, A. Del Bue, L. Furia, M. Faretta, and A. Diaspro, “Live-cell 3D super-resolution imaging in thick biological samples,” Nat. Methods8(12), 1047–1049 (2011). [CrossRef] [PubMed]
  6. C. J. Sheppard and T. Wilson, “Depth of field in the scanning microscope,” Opt. Lett.3(3), 115–117 (1978). [CrossRef] [PubMed]
  7. A. Rohrbach, “Artifacts resulting from imaging in scattering media: a theoretical prediction,” Opt. Lett.34(19), 3041–3043 (2009). [CrossRef] [PubMed]
  8. V. Ntziachristos, “Going deeper than microscopy: the optical imaging frontier in biology,” Nat. Methods7(8), 603–614 (2010). [CrossRef] [PubMed]
  9. J. Huisken and D. Y. Stainier, “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM),” Opt. Lett.32(17), 2608–2610 (2007). [CrossRef] [PubMed]
  10. R. Tomer, K. Khairy, F. Amat, and P. J. Keller, “Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy,” Nat. Methods9(7), 755–763 (2012). [CrossRef] [PubMed]
  11. U. Krzic, S. Gunther, T. E. Saunders, S. J. Streichan, and L. Hufnagel, “Multiview light-sheet microscope for rapid in toto imaging,” Nat. Methods9(7), 730–733 (2012). [CrossRef] [PubMed]
  12. T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods8(5), 417–423 (2011). [CrossRef] [PubMed]
  13. F. O. Fahrbach, P. Simon, and A. Rohrbach, “Microscopy with self-reconstructing beams,” Nat. Photonics4(11), 780–785 (2010). [CrossRef]
  14. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990). [CrossRef] [PubMed]
  15. A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence exitation and related thechniques in biological microscopy,” Q. Rev. Biophys.15, 1–70 (2006).
  16. J. Palero, S. I. C. O. Santos, D. Artigas, and P. Loza-Alvarez, “A simple scanless two-photon fluorescence microscope using selective plane illumination,” Opt. Express18(8), 8491–8498 (2010). [CrossRef] [PubMed]
  17. F. Cella Zanacchi, Z. Lavagnino, E. Ronzitti, and A. Diaspro, “Two-photon fluorescence excitation within a light sheet based microscopy architecture,” Proc. SPIE7903, 7903–7906 (2011). [CrossRef]
  18. T. V. Truong, W. Supatto, D. S. Koos, J. M. Choi, and S. E. Fraser, “Deep and fast live imaging with two-photon scanned light-sheet microscopy,” Nat. Methods8(9), 757–760 (2011). [CrossRef] [PubMed]
  19. W. Supatto, T. V. Truong, D. De’ Barrel, and E. Beaurepair “Advances in multiphoton microscopy for imaging embryos” Curr Opinion Gen. Dev (21) 538–548 (2011)
  20. P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A23(12), 3139–3149 (2006). [CrossRef] [PubMed]
  21. K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum.78(2), 023705 (2007). [CrossRef] [PubMed]
  22. A. N. Yaroslavsky, P. C. Schulze, I. V. Yaroslavsky, R. Schober, F. Ulrich, and H. J. Schwarzmaier, “Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range,” Phys. Med. Biol.47(12), 2059–2073 (2002). [CrossRef] [PubMed]
  23. J. R. Mourant, J. P. Freyer, A. H. Hielscher, A. A. Eick, D. Shen, and T. M. Johnson, “Mechanisms of light scattering from biological cells relevant to noninvasive optical-tissue diagnostics,” Appl. Opt.37(16), 3586–3593 (1998). [CrossRef] [PubMed]
  24. W. Cheong, S. Prahl, and A. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron.26(12), 2166–2185 (1990). [CrossRef]
  25. S. Prahl, Mie Scattering Calculator, http://omlc.ogi.edu/calc/mie_calc.html
  26. J. Debnath, S. K. Muthuswamy, and J. S. Brugge, “Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures,” Methods30(3), 256–268 (2003). [CrossRef] [PubMed]
  27. B. E. A. Saleh and M. C. Teich, Fundamentals of Photonics (Wiley & Sons, 1991), Chap. 3.
  28. J. G. Ritter, R. Veith, J.-P. Siebrasse, and U. Kubitscheck, “High-contrast single-particle tracking by selective focal plane illumination microscopy,” Opt. Express16(10), 7142–7152 (2008). [CrossRef] [PubMed]
  29. J. Ying, F. Liu, and R. R. Alfano, “Spatial distribution of two-photon-excited fluorescence in scattering media,” Appl. Opt.38(1), 224–229 (1999). [CrossRef] [PubMed]
  30. P. F. Mullaney and P. N. Dean, “The small angle light scattering of biological cells. Theoretical considerations,” Biophys. J.10(8), 764–772 (1970). [CrossRef] [PubMed]
  31. J. Ying, F. Liu, and R. R. Alfano, “Effect of scattering on nonlinear optical scanning microscopy imaging of highly scattering media,” Appl. Opt.39(4), 509–514 (2000). [CrossRef] [PubMed]
  32. C. J. Engelbrecht and E. H. K. Stelzer. “Resolution enhancement in a light-sheet-based microscope (SPIM).” Opt Lett. 10, 1477–1479 (2006)
  33. J. T. Bushberg, J. A. Seibert, E. M. Leidholt, Jr., and J. M. Boone, The Essential Physics of Medical Imaging (Lippincott Williams & Wilkins, 2006) Chap. 10.

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