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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 6, Iss. 9 — Oct. 3, 2011

Maximizing fluorescence collection efficiency in multiphoton microscopy

Joseph P. Zinter and Michael J. Levene  »View Author Affiliations


Optics Express, Vol. 19, Issue 16, pp. 15348-15362 (2011)
http://dx.doi.org/10.1364/OE.19.015348


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Abstract

Understanding fluorescence propagation through a multiphoton microscope is of critical importance in designing high performance systems capable of deep tissue imaging. Optical models of a scattering tissue sample and the Olympus 20X 0.95NA microscope objective were used to simulate fluorescence propagation as a function of imaging depth for physiologically relevant scattering parameters. The spatio-angular distribution of fluorescence at the objective back aperture derived from these simulations was used to design a simple, maximally efficient post-objective fluorescence collection system. Monte Carlo simulations corroborated by data from experimental tissue phantoms demonstrate collection efficiency improvements of 50% – 90% over conventional, non-optimized fluorescence collection geometries at large imaging depths. Imaging performance was verified by imaging layer V neurons in mouse cortex to a depth of 850 μm.

© 2011 OSA

OCIS Codes
(180.0180) Microscopy : Microscopy
(180.2520) Microscopy : Fluorescence microscopy
(220.0220) Optical design and fabrication : Optical design and fabrication
(220.4830) Optical design and fabrication : Systems design
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:
Microscopy

History
Original Manuscript: June 10, 2011
Revised Manuscript: July 11, 2011
Manuscript Accepted: July 11, 2011
Published: July 26, 2011

Virtual Issues
Vol. 6, Iss. 9 Virtual Journal for Biomedical Optics

Citation
Joseph P. Zinter and Michael J. Levene, "Maximizing fluorescence collection efficiency in multiphoton microscopy," Opt. Express 19, 15348-15362 (2011)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-19-16-15348


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References

  1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990). [CrossRef] [PubMed]
  2. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003). [CrossRef] [PubMed]
  3. C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13(3), 481–491 (1996). [CrossRef]
  4. E. Beaurepaire, M. Oheim, and J. Mertz, “Ultra-deep two-photon fluorescence excitation in turbid media,” Opt. Commun. 188(1–4), 25–29 (2001). [CrossRef]
  5. P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 microm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett. 28(12), 1022–1024 (2003). [CrossRef] [PubMed]
  6. A. Leray, C. Odin, E. Huguet, F. Amblard, and Y. Le Grand, “Spatially distributed two-photon excitation fluorescence in scattering media: Experiments and time-resolved Monte Carlo simulations,” Opt. Commun. 272(1), 269–278 (2007). [CrossRef]
  7. A. Leray, C. Odin, and Y. Le Grand, “Out-of-focus fluorescence collection in two-photon microscopy of scattering media,” Opt. Commun. 281(24), 6139–6144 (2008). [CrossRef]
  8. P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A 23(12), 3139–3149 (2006). [CrossRef] [PubMed]
  9. D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009). [CrossRef] [PubMed]
  10. S. G. Parra, T. H. Chia, J. P. Zinter, and M. J. Levene, “Multiphoton microscopy of cleared mouse organs,” J. Biomed. Opt. 15(3), 036017 (2010). [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(1), 29–37 (2001). [CrossRef] [PubMed]
  12. V. Tuchin, Tissue Optics, 2.ed., (SPIE, Bellingham, Washington, 2000).
  13. E. Beaurepaire and J. Mertz, “Epifluorescence collection in two-photon microscopy,” Appl. Opt. 41(25), 5376–5382 (2002). [CrossRef] [PubMed]
  14. Y. Le Grand, A. Leray, T. Guilbert, and C. Odin, “Non-descanned versus descanned epifluorescence collection in two-photon microscopy: Experiments and Monte Carlo simulations,” Opt. Commun. 281(21), 5480–5486 (2008). [CrossRef]

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