Abstract

Achieving a greater imaging depth with two-photon fluorescence microscopy (TPFM) is mainly limited by out-of-focus fluorescence generated from both ballistic and scattered light excitation. We report on an improved signal-to-noise ratio (SNR) in a highly scattering medium as demonstrated by analytical simulation and experiments for TPFM. Our technique is based on out-of-focus rejection using a confocal pinhole. We improved the SNR by introducing the pinhole in the collection beam path. Using the radiative transfer theory and the ray-optics approach, we analyzed the effects of different sizes of pinholes on the generation of the fluorescent signal in the TPFM system. The analytical simulation was evaluated by comparing its results with the experimental results in a scattering medium. In a combined confocal pinhole and two-photon microscopy system, the imaging depth limit of approximately 5 scattering mean free paths (MFP) was found to have improved to 6.2 MFP.

© 2012 OSA

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. S. Paddock, “Tech.Sight. Optical sectioning--slices of life,” Science295(5558), 1319–1321 (2002).
    [CrossRef] [PubMed]
  2. J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods2(12), 920–931 (2005).
    [CrossRef] [PubMed]
  3. F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods2(12), 932–940 (2005).
    [CrossRef] [PubMed]
  4. 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]
  5. J. G. White and W. B. Amos, “Confocal microscopy comes of age,” Nature328(6126), 183–184 (1987).
    [CrossRef]
  6. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
    [CrossRef] [PubMed]
  7. V. V. Tuchin, Tissue optics: Light scattering methods and instruments for medical diagnosis (Springer-Verlag, 2006).
  8. J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer, 2006).
  9. 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]
  10. 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]
  11. Y. L. 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).
  12. C. J. Engelbrecht, W. Göbel, and F. Helmchen, “Enhanced fluorescence signal in nonlinear microscopy through supplementary fiber-optic light collection,” Opt. Express17(8), 6421–6435 (2009).
    [CrossRef] [PubMed]
  13. R. Gauderon, P. B. Lukins, and C. J. Sheppard, “Effect of a confocal pinhole in two-photon microscopy,” Microsc. Res. Tech.47(3), 210–214 (1999).
    [CrossRef] [PubMed]
  14. A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J.94(4), 1449–1458 (2008).
    [CrossRef] [PubMed]
  15. A. Leray, C. Odin, and Y. L. Grand, “Out-of-focus fluorescence collection in two-photon microscopy of scattering media,” Opt. Commun.281(24), 6139–6144 (2008).
    [CrossRef]
  16. S. A. Prahl, M. Keijzer, S. L. Jacques, and A. J. Welch, “A Monte Carlo model of light propagation in tissue,” Proc. SPIE IS 5, 102–111 (1989).
  17. L. Wang, S. L. Jacques, and L. Zheng, “MCML-Monte Carlo modeling of light transport in multi-layered tissues,” Comput. Methods Programs Biomed.47(2), 131–146 (1995).
    [CrossRef] [PubMed]
  18. Z. Wang, Z. Zhang, Z. Xu, and Q. Lin, “Space-time profile of an ultrashort pulsed Gaussian beam,” IEEE J. Quant. Electron.33(4), 566–573 (1997).
    [CrossRef]
  19. M. A. Porras, “Ultrashort pulsed Gaussian light beams,” Phys. Rev. E58(1), 1086–1093 (1998).
    [CrossRef]
  20. X. Fu, H. Guo, W. Hu, and S. Yu, “Spatial nonparaxial correction of the ultrashort pulsed beam propagation in free space,” Phys. Rev. E Stat. Nonlin. Soft Matter Phys.65(5), 056611 (2002).
    [CrossRef] [PubMed]
  21. 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. B13(3), 481–491 (1996).
    [CrossRef]
  22. P. Theer, “On the fundamental imaging-depth limit in two-photon microscopy,” Dissertation, University of Heidelberg (2004).
  23. J. W. McLean, J. D. Freeman, and R. E. Walker, “Beam spread function with time dispersion,” Appl. Opt.37(21), 4701–4711 (1998).
    [CrossRef] [PubMed]
  24. A. K. Dunn, V. P. Wallace, M. Coleno, M. W. Berns, and B. J. Tromberg, “Influence of optical properties on two-photon fluorescence imaging in turbid samples,” Appl. Opt.39(7), 1194–1201 (2000).
    [CrossRef] [PubMed]
  25. M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods111(1), 29–37 (2001).
    [CrossRef] [PubMed]
  26. E. Beaurepaire and J. Mertz, “Epifluorescence collection in two-photon microscopy,” Appl. Opt.41(25), 5376–5382 (2002).
    [CrossRef] [PubMed]
  27. J. D. Jackson, “Classical Electrodynamics,” John Wiley & Sons, Inc., New York, (1999).
  28. C. Matzler, Matlab Functions for Mie Scattering and Absorption (Institut fur Angewandte Physik: University of Heidelberg 2004).
  29. J. P. Zinter and M. J. Levene, “Maximizing fluorescence collection efficiency in multiphoton microscopy,” Opt. Express19(16), 15348–15362 (2011).
    [CrossRef] [PubMed]
  30. W. W. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc.169(3), 391–405 (1993).
    [CrossRef]
  31. 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]

2011

2010

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

2008

A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J.94(4), 1449–1458 (2008).
[CrossRef] [PubMed]

A. Leray, C. Odin, and Y. L. Grand, “Out-of-focus fluorescence collection in two-photon microscopy of scattering media,” Opt. Commun.281(24), 6139–6144 (2008).
[CrossRef]

Y. L. 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).

2006

2005

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods2(12), 920–931 (2005).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods2(12), 932–940 (2005).
[CrossRef] [PubMed]

2003

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]

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]

2002

S. Paddock, “Tech.Sight. Optical sectioning--slices of life,” Science295(5558), 1319–1321 (2002).
[CrossRef] [PubMed]

X. Fu, H. Guo, W. Hu, and S. Yu, “Spatial nonparaxial correction of the ultrashort pulsed beam propagation in free space,” Phys. Rev. E Stat. Nonlin. Soft Matter Phys.65(5), 056611 (2002).
[CrossRef] [PubMed]

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

2001

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods111(1), 29–37 (2001).
[CrossRef] [PubMed]

2000

1999

R. Gauderon, P. B. Lukins, and C. J. Sheppard, “Effect of a confocal pinhole in two-photon microscopy,” Microsc. Res. Tech.47(3), 210–214 (1999).
[CrossRef] [PubMed]

1998

1997

Z. Wang, Z. Zhang, Z. Xu, and Q. Lin, “Space-time profile of an ultrashort pulsed Gaussian beam,” IEEE J. Quant. Electron.33(4), 566–573 (1997).
[CrossRef]

1996

1995

L. Wang, S. L. Jacques, and L. Zheng, “MCML-Monte Carlo modeling of light transport in multi-layered tissues,” Comput. Methods Programs Biomed.47(2), 131–146 (1995).
[CrossRef] [PubMed]

1993

W. W. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc.169(3), 391–405 (1993).
[CrossRef]

1990

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

1987

J. G. White and W. B. Amos, “Confocal microscopy comes of age,” Nature328(6126), 183–184 (1987).
[CrossRef]

Amos, W. B.

J. G. White and W. B. Amos, “Confocal microscopy comes of age,” Nature328(6126), 183–184 (1987).
[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. Methods111(1), 29–37 (2001).
[CrossRef] [PubMed]

Berns, M. W.

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. Methods111(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. Methods111(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]

Coleno, M.

Conchello, J. A.

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods2(12), 920–931 (2005).
[CrossRef] [PubMed]

Cremer, C.

W. W. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc.169(3), 391–405 (1993).
[CrossRef]

Denk, W.

Dunn, A. K.

Engelbrecht, C. J.

Freeman, J. D.

Fu, X.

X. Fu, H. Guo, W. Hu, and S. Yu, “Spatial nonparaxial correction of the ultrashort pulsed beam propagation in free space,” Phys. Rev. E Stat. Nonlin. Soft Matter Phys.65(5), 056611 (2002).
[CrossRef] [PubMed]

Gauderon, R.

R. Gauderon, P. B. Lukins, and C. J. Sheppard, “Effect of a confocal pinhole in two-photon microscopy,” Microsc. Res. Tech.47(3), 210–214 (1999).
[CrossRef] [PubMed]

Göbel, W.

Grand, Y. L.

Y. L. 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).

A. Leray, C. Odin, and Y. L. Grand, “Out-of-focus fluorescence collection in two-photon microscopy of scattering media,” Opt. Commun.281(24), 6139–6144 (2008).
[CrossRef]

Guilbert, T.

Y. L. 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).

Guo, H.

X. Fu, H. Guo, W. Hu, and S. Yu, “Spatial nonparaxial correction of the ultrashort pulsed beam propagation in free space,” Phys. Rev. E Stat. Nonlin. Soft Matter Phys.65(5), 056611 (2002).
[CrossRef] [PubMed]

Hasan, M. T.

Hell, W. W.

W. W. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc.169(3), 391–405 (1993).
[CrossRef]

Helmchen, F.

Hu, W.

X. Fu, H. Guo, W. Hu, and S. Yu, “Spatial nonparaxial correction of the ultrashort pulsed beam propagation in free space,” Phys. Rev. E Stat. Nonlin. Soft Matter Phys.65(5), 056611 (2002).
[CrossRef] [PubMed]

Jacques, S. L.

L. Wang, S. L. Jacques, and L. Zheng, “MCML-Monte Carlo modeling of light transport in multi-layered tissues,” Comput. Methods Programs Biomed.47(2), 131–146 (1995).
[CrossRef] [PubMed]

Leray, A.

A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J.94(4), 1449–1458 (2008).
[CrossRef] [PubMed]

A. Leray, C. Odin, and Y. L. Grand, “Out-of-focus fluorescence collection in two-photon microscopy of scattering media,” Opt. Commun.281(24), 6139–6144 (2008).
[CrossRef]

Y. L. 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).

Levene, M. J.

J. P. Zinter and M. J. Levene, “Maximizing fluorescence collection efficiency in multiphoton microscopy,” Opt. Express19(16), 15348–15362 (2011).
[CrossRef] [PubMed]

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]

Lichtman, J. W.

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods2(12), 920–931 (2005).
[CrossRef] [PubMed]

Lillis, K.

A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J.94(4), 1449–1458 (2008).
[CrossRef] [PubMed]

Lin, Q.

Z. Wang, Z. Zhang, Z. Xu, and Q. Lin, “Space-time profile of an ultrashort pulsed Gaussian beam,” IEEE J. Quant. Electron.33(4), 566–573 (1997).
[CrossRef]

Lukins, P. B.

R. Gauderon, P. B. Lukins, and C. J. Sheppard, “Effect of a confocal pinhole in two-photon microscopy,” Microsc. Res. Tech.47(3), 210–214 (1999).
[CrossRef] [PubMed]

McLean, J. W.

Mertz, J.

A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J.94(4), 1449–1458 (2008).
[CrossRef] [PubMed]

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. Methods111(1), 29–37 (2001).
[CrossRef] [PubMed]

Odin, C.

A. Leray, C. Odin, and Y. L. Grand, “Out-of-focus fluorescence collection in two-photon microscopy of scattering media,” Opt. Commun.281(24), 6139–6144 (2008).
[CrossRef]

Y. L. 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).

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. Methods111(1), 29–37 (2001).
[CrossRef] [PubMed]

Paddock, S.

S. Paddock, “Tech.Sight. Optical sectioning--slices of life,” Science295(5558), 1319–1321 (2002).
[CrossRef] [PubMed]

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]

Porras, M. A.

M. A. Porras, “Ultrashort pulsed Gaussian light beams,” Phys. Rev. E58(1), 1086–1093 (1998).
[CrossRef]

Reiner, G.

W. W. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc.169(3), 391–405 (1993).
[CrossRef]

Sheppard, C. J.

R. Gauderon, P. B. Lukins, and C. J. Sheppard, “Effect of a confocal pinhole in two-photon microscopy,” Microsc. Res. Tech.47(3), 210–214 (1999).
[CrossRef] [PubMed]

Stelzer, E. H. K.

W. W. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc.169(3), 391–405 (1993).
[CrossRef]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Theer, P.

Tromberg, B. J.

Walker, R. E.

Wallace, V. P.

Wang, L.

L. Wang, S. L. Jacques, and L. Zheng, “MCML-Monte Carlo modeling of light transport in multi-layered tissues,” Comput. Methods Programs Biomed.47(2), 131–146 (1995).
[CrossRef] [PubMed]

Wang, Z.

Z. Wang, Z. Zhang, Z. Xu, and Q. Lin, “Space-time profile of an ultrashort pulsed Gaussian beam,” IEEE J. Quant. Electron.33(4), 566–573 (1997).
[CrossRef]

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. B13(3), 481–491 (1996).
[CrossRef]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

White, J. G.

J. G. White and W. B. Amos, “Confocal microscopy comes of age,” Nature328(6126), 183–184 (1987).
[CrossRef]

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]

Xu, C.

Xu, Z.

Z. Wang, Z. Zhang, Z. Xu, and Q. Lin, “Space-time profile of an ultrashort pulsed Gaussian beam,” IEEE J. Quant. Electron.33(4), 566–573 (1997).
[CrossRef]

Yu, S.

X. Fu, H. Guo, W. Hu, and S. Yu, “Spatial nonparaxial correction of the ultrashort pulsed beam propagation in free space,” Phys. Rev. E Stat. Nonlin. Soft Matter Phys.65(5), 056611 (2002).
[CrossRef] [PubMed]

Zhang, Z.

Z. Wang, Z. Zhang, Z. Xu, and Q. Lin, “Space-time profile of an ultrashort pulsed Gaussian beam,” IEEE J. Quant. Electron.33(4), 566–573 (1997).
[CrossRef]

Zheng, L.

L. Wang, S. L. Jacques, and L. Zheng, “MCML-Monte Carlo modeling of light transport in multi-layered tissues,” Comput. Methods Programs Biomed.47(2), 131–146 (1995).
[CrossRef] [PubMed]

Zinter, J. P.

J. P. Zinter and M. J. Levene, “Maximizing fluorescence collection efficiency in multiphoton microscopy,” Opt. Express19(16), 15348–15362 (2011).
[CrossRef] [PubMed]

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.

Biophys. J.

A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J.94(4), 1449–1458 (2008).
[CrossRef] [PubMed]

Comput. Methods Programs Biomed.

L. Wang, S. L. Jacques, and L. Zheng, “MCML-Monte Carlo modeling of light transport in multi-layered tissues,” Comput. Methods Programs Biomed.47(2), 131–146 (1995).
[CrossRef] [PubMed]

IEEE J. Quant. Electron.

Z. Wang, Z. Zhang, Z. Xu, and Q. Lin, “Space-time profile of an ultrashort pulsed Gaussian beam,” IEEE J. Quant. Electron.33(4), 566–573 (1997).
[CrossRef]

J. Biomed. Opt.

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. Microsc.

W. W. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc.169(3), 391–405 (1993).
[CrossRef]

J. Neurosci. Methods

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods111(1), 29–37 (2001).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A

J. Opt. Soc. Am. B

Microsc. Res. Tech.

R. Gauderon, P. B. Lukins, and C. J. Sheppard, “Effect of a confocal pinhole in two-photon microscopy,” Microsc. Res. Tech.47(3), 210–214 (1999).
[CrossRef] [PubMed]

Nat. Biotechnol.

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]

Nat. Methods

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods2(12), 920–931 (2005).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods2(12), 932–940 (2005).
[CrossRef] [PubMed]

Nature

J. G. White and W. B. Amos, “Confocal microscopy comes of age,” Nature328(6126), 183–184 (1987).
[CrossRef]

Opt. Commun.

Y. L. 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).

A. Leray, C. Odin, and Y. L. Grand, “Out-of-focus fluorescence collection in two-photon microscopy of scattering media,” Opt. Commun.281(24), 6139–6144 (2008).
[CrossRef]

Opt. Express

Opt. Lett.

Phys. Rev. E

M. A. Porras, “Ultrashort pulsed Gaussian light beams,” Phys. Rev. E58(1), 1086–1093 (1998).
[CrossRef]

Phys. Rev. E Stat. Nonlin. Soft Matter Phys.

X. Fu, H. Guo, W. Hu, and S. Yu, “Spatial nonparaxial correction of the ultrashort pulsed beam propagation in free space,” Phys. Rev. E Stat. Nonlin. Soft Matter Phys.65(5), 056611 (2002).
[CrossRef] [PubMed]

Science

S. Paddock, “Tech.Sight. Optical sectioning--slices of life,” Science295(5558), 1319–1321 (2002).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Other

V. V. Tuchin, Tissue optics: Light scattering methods and instruments for medical diagnosis (Springer-Verlag, 2006).

J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer, 2006).

S. A. Prahl, M. Keijzer, S. L. Jacques, and A. J. Welch, “A Monte Carlo model of light propagation in tissue,” Proc. SPIE IS 5, 102–111 (1989).

P. Theer, “On the fundamental imaging-depth limit in two-photon microscopy,” Dissertation, University of Heidelberg (2004).

J. D. Jackson, “Classical Electrodynamics,” John Wiley & Sons, Inc., New York, (1999).

C. Matzler, Matlab Functions for Mie Scattering and Absorption (Institut fur Angewandte Physik: University of Heidelberg 2004).

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.


Figures (11)

Fig. 1
Fig. 1

Simulated spatial distribution of ballistic (left) and scattered (right) light intensity along the depth z (z = 0, 0.4, 0.8, 1.2, 1.6, 2, and 2.4 mm) for λ = 810 nm, μs = 25 cm−1, NA = 0.9, τ0 = 140 fs, and g = 0.9. The timescales of ballistic and scattered cases are different, −1 ps to 3 ps and −6 ps to 6 ps, respectively.

Fig. 2
Fig. 2

Schematic diagram of TPEF collection path with pinhole. The figure only shows the on-axis terms. fo and ft are the focal length of the objective and tube lens, ro and rt are the radii of the objective and tube lens, wd is the working distance of the objective lens, ro is the objective front aperture radius, θp is the angular acceptable range according to the radius of pinhole rp, and θ is the maximum acceptance angle at each depth.

Fig. 3
Fig. 3

(a) A TPEF collection from an arbitrarily positioned fluorescence source. The solid angle Ω depends on the acceptable area radius ra, fluorescence distance R, and the off-axis angle γ. (b) A solid angle of the ellipse (left) and its identical solid angle of the circle (right). We assume that the areas of the ellipse and circle are identical; the solid angle is also the same (ra rγ = rm2).

Fig. 4
Fig. 4

Spatial distribution for collection efficiency of 20-μm pinhole (left) and non-pinhole (2500-μm)(right) in non-scattering medium corresponding to an arbitrarily positioned fluorescence source. Data were plotted on a semi-logarithmic scale.

Fig. 5
Fig. 5

Spatially distributed total collection efficiency of 20-μm pinhole (left) and non-pinhole (right) in scattering medium. The scattering coefficient at the fluorescence wavelength is 42 cm−1. Data were plotted on a semi-logarithmic scale.

Fig. 6
Fig. 6

Axial profiles of the collection efficiency of 20-μm (blue) and non-pinhole (red) for a focusing depth set at 2000 um. The scattering mean free path MFP at excitation and TPEF wavelength are also reported on the upper x-axis. The corresponding ratios of non-pinhole and 20 um pinhole profiles are also provided (dotted line). The scales are the same for individual collection efficiency profiles and a ratio.

Fig. 7
Fig. 7

Semi-logarithmic representation of excitation (black line) and TPEF collection of 20-μm pinhole (red line) and non-pinhole (blue line) pinholes for the same excitation condition as that for Fig. 1 and at μf = 42cm−1 the fluorescence wavelength of 585 nm. The corresponding ratio of the 20-μm and non-pinhole profile is also provided for comparison (dotted green line). Data were normalized by excitation intensity at the surface (z = 0).

Fig. 8
Fig. 8

Semi-logarithmic plot of measured and simulated axial TPEF intensity according to 20-, 50- and 150-μm-diameter pinholes (blue asterisk, red christcross, and black cross, respectively). Measured data were normalized by the surface intensity of the 20-μm pinhole and simulation data were normalized by the surface maximum value of the 20-μm pinhole. The excitation simulation was run with NA 0.5 and axial TPEF intensity was summed with 20 μm to compensate for the 20-μm thickness of the experiment.

Fig. 9
Fig. 9

Signal-to-noise ratio simulated with 20 μm (blue square) and non-pinhole (red circle) pinholes as a function of scattering MFP μsz. The corresponding SNR for excitation is also plotted for comparison (black triangle). The constraint of imaging depth is assumed to be fallen at SNR = 1

Fig. 10
Fig. 10

Space-time pulse shape observed at the surface z = 0. r is the distance from the optical axis and t is the relative time. λ0 is 810 nm and τ0 is 100 fs, NA is 1 and focus depth is 500 μm.

Fig. 11
Fig. 11

Averaged pulse width (black line) and each pulse width observed at each lateral r position (0, 200, 400, 600 μm) at the surface z = 0. The red line is for r = 0 μm, blue line is for r = 200 μm, pink line is for r = 400 μm, and the cyan one is for r = 600 μm from the optical axis.

Equations (28)

Equations on this page are rendered with MathJax. Learn more.

I(r,z,t)= P 0 2 w 0 2 w (z) 2 exp( 2 (t r 2 2cR(z) z c ) 2 τ 0 2 )exp(2( r 2 w (z) 2 + r 4 w (z) 4 1 ( ω 0 τ 0 ) 2 )),
Q(r,z,t) v q(r,t)dtdV,q(r,t)= δC(r,t) I 2 (r,t) hcλ 1 τ 0 F 2 ,
I t (r,z,t)= I b (r,z,t)+ I s (r,z,t),
I b (r,z,t)=I(r,z,t)exp( μ s z).
I s (r,z,t)= P 0 2 1exp( μ s z) π w s (z) 2 exp( 2 r 2 w s (z) 2 )exp( 2 t 2 τ s (z) 2 ),
I ' s (r,z,t)= I s (r,z,t')f(tt')dt' .
tan 1 ( r o r z )θ(r,z) tan 1 ( r o r z ),
tan 1 ( r f o tan( θ p ) f o z )θ(r,z) tan 1 ( r+ f o tan( θ p ) f o z ),
η ns (0,z)= Ω onaxis (z) 4π = 1 2 (1cos(θ(0,z))),
η ns (r,z)= Ω m (z) 4π = 1 2 (1cos(arctan( r m R )))= 1 2 (1cos(arctan( r a r γ cosγ z ))),
G(r,z)= Q 4πD ( 1 r 2 + ( z 0 z) 2 1 r 2 + ( z 0 +z) 2 ),
F(r,z)= D z G(r,z) | z=0 = Q 2π z 0 ( r 2 + z 0 2 ) 3/2 .
F p (r, z 0 )= D z G(r,z) Ω m (r,z=0) 2π | z=0 = Q 2π z 0 ( r 2 + z 0 2 ) 3/2 Ω m (r,0) 2π ,
η ns (0, z 0 )= Ω(0, z 0 ) 4π F p (r, z 0 )2πrdr ,
η s (r, z 0 )= Ω(r, z 0 ) 4π F p (r, z 0 )2πrdr = 1 2 (1cos( tan 1 ( r sm z 0 cosγ))) F p (r, z 0 )2πrdr ,
η(r,z)= η ns exp( μ f R)+ η s (1exp( μ f R)).
F s η(r,z) I t (r,z)2πrdr.
( 2 1 c 2 2 t 2 )E( r ,z,t)=0,
[ 2 + 2 t 2 +2ik(ω) z ] U ˜ ( r ,z,ω)=0,
E( r ,z,t)= 1 2π U ˜ ( r ,z,ω)exp(iωτ)dω .
| z U ˜ ( r ,z,ω) || k(ω) U ˜ ( r ,z,ω) |,
| 2 z 2 U ˜ ( r ,z,ω) || k(ω) z U ˜ ( r ,z,ω) |,
[ 2 +2ik(ω) z ] U ˜ ( r ,z,ω)=0,
U ˜ ( r ,z,ω)= i z 0 q(z) exp(ik r 2 2q(z) )P(ω),
E( r ,z,τ)= i z 0 q(z) P(τ'), P(τ')= 1 2π P(ω)exp(iω( r 2 2qc τ))dω
P(τ)=exp( τ 2 τ 0 2 ){exp(i ω 0 t)iIm[exp(i ω 0 τ)erfc( τ 0 2 +i τ τ 0 )]},
E( r ,z,τ)= P 0 w 0 w(z) exp( (t r 2 2cR(z) z c ) 2 τ 0 2 )exp( r 2 w (z) 2 + r 4 w (z) 4 1 ( ω 0 τ 0 ) 2 ) ×exp(i ω 0 (t z c r 2 2cR(z) )(1 2 r 2 w (z) 2 1 ω 0 τ 0 ))exp(iξ(z)),
I( r ,z,t)= | E( r ,z,t) | 2 = P 0 w 0 2 w (z) 2 exp( 2 (t r 2 2cR(z) z c ) 2 τ 0 2 )exp( 2 r 2 w (z) 2 + 2 r 4 w (z) 4 1 ( ω 0 τ 0 ) 2 ).

Metrics