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

PDF Article

References

  • View by:
  • |
  • |
  • |

  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]

2010 (1)

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]

2009 (1)

2008 (2)

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]

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]

2007 (1)

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]

2006 (1)

2003 (2)

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]

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]

2002 (1)

2001 (2)

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]

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]

1996 (1)

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Amblard, F.

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]

Beaurepaire, E.

E. Beaurepaire and J. Mertz, “Epifluorescence collection in two-photon microscopy,” Appl. Opt. 41(25), 5376–5382 (2002).
[CrossRef] [PubMed]

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]

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]

Chaigneau, E.

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]

Charpak, S.

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]

Chia, T. H.

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]

Denk, W.

Durst, M. E.

Guilbert, T.

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]

Hasan, M. T.

Huguet, E.

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]

Kobat, D.

Le Grand, Y.

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]

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]

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]

Leray, A.

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]

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]

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]

Levene, M. J.

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]

Mertz, J.

E. Beaurepaire and J. Mertz, “Epifluorescence collection in two-photon microscopy,” Appl. Opt. 41(25), 5376–5382 (2002).
[CrossRef] [PubMed]

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]

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]

Nishimura, N.

Odin, C.

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]

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]

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]

Oheim, M.

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]

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]

Parra, S. G.

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]

Schaffer, C. B.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Theer, P.

Webb, W. W.

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]

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]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Williams, R. M.

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]

Wong, A. W.

Xu, C.

Zinter, J. P.

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]

Zipfel, W. R.

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]

Appl. Opt. (1)

J. Biomed. Opt. (1)

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]

J. Neurosci. Methods (1)

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]

J. Opt. Soc. Am. A (1)

J. Opt. Soc. Am. B (1)

Nat. Biotechnol. (1)

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]

Opt. Commun. (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]

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]

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]

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]

Opt. Express (1)

Opt. Lett. (1)

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Other (1)

V. Tuchin, Tissue Optics, 2.ed., (SPIE, Bellingham, Washington, 2000).

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Metrics