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

Full Article  |  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)

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, and Y. Le Grand, “Out-of-focus fluorescence collection in two-photon microscopy of scattering media,” Opt. Commun. 281(24), 6139–6144 (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)

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]

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]

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]

Nishimura, N.

Odin, C.

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, and Y. Le Grand, “Out-of-focus fluorescence collection in two-photon microscopy of scattering media,” Opt. Commun. 281(24), 6139–6144 (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.


Figures (8)

Fig. 1
Fig. 1

Propagation of isotropically emitted fluorescent photons through a microscope objective for a non-scattering case (left) and scattering case (right). In the non-scattering case, photons that fall within the objective NA (illustrated as dashed lines) are collected, and emerge from the OBA in a collimated fashion. In the scattering case, photon trajectories are determined by the scattering properties of the sample. Scattering increases the total number of collected photons since photons that normally would not be collected can be scattered towards and enter the OFA. Scattered fluorescence entering the OFA results in a spatio-angular distribution of photons emerging from the OBA.

Fig. 2
Fig. 2

Zemax schematic (left), and SolidWorks renderings (middle and right) of the Olympus 20X 0.95NA objective used for Monte Carlo simulations and opto-mechanical design.

Fig. 3
Fig. 3

Zemax Monte Carlo ray trace. a) Scattered photons (blue rays) propagating through a simulated scattering medium and entering the OFA of the Olympus 20X 0.95Na objective. b) Trimetric wireframe ray trace of the unfolded optimized post-objective fluorescence collection optical system. Rays exiting the scattering medium (left) travel through and emerge from the objective with a angular distribution. Collection lenses (Lens 1 and Lens 2) guide this fluorescence onto the PMT photocathode (right). Each ray segment is mapped to a different color. In both a) and b) rays exiting the optical system have been removed for clarity.

Fig. 4
Fig. 4

Mechanical renderings of the optimized fluorescence collection system and detector housing assembly. a) Cutaway model of the microscope objective, detector housing, and H7422P-40mod GaAsP PMT. Green lines represent fluorescence propagation from the OBA to photocathode. Inset illustrations (b and c) show the closed detector housing assembly (b), and the same assembly with the top and one detector removed (c). SM1 optical tubes used to house the lenses and band pass filters have been removed to better illustrate function.

Fig. 5
Fig. 5

Zemax Monte Carlo simulations for fluorescence propagation through the Olympus 20X 0.95NA microscope objective. a) Collected fraction of total fluorescence emitted from an isotropic source as a function of depth for emission scattering lengths of 50, 75, and 100 μm, normalized to emission scattering length. Solid color lines represent photon flux incident at the OFA. Dashed color lines represent the transmitted intensity measured at the OBA for the three scattering cases. The horizontal dashed line indicates the collected and transmitted fluorescence without scattering assuming an NA = 0.95. b) Collected fraction of fluorescence emerging from the OBA as a function of collection angle for emission scattering lengths of 50, 75, and 100 μm, for surface (z = 0 μm) and deep (z = 1000 μm) imaging. Vertical dashed lines correspond to a standard 23 mm and custom 38 mm clear aperture at the evaluation location described in the text.

Fig. 6
Fig. 6

Monte Carlo simulations of optimized and non-optimized fluorescence collection system performance. a) Unfolded optimized fluorescence collection system. b) Fraction of total fluorescence collected for the optimized housing design as a function of imaging depth evaluated at each system aperture. c) Comparison of fluorescence striking the PMT photocathodes for optimized and conventional geometries, normalized to the fluorescence emerging from the OBA. See text for description of geometrical configurations. Data in this figure pertains to a sample with an emission scattering length of 100 μm at 520 nm.

Fig. 7
Fig. 7

Experimental tissue phantom performance. a) Fluorescence signal as a function of depth for tissue phantoms with emission scattering lengths of 50, 75, and 100 μm at 520 nm (corresponding to excitation scattering lengths of 100, 150, and 200 μm, respectively, at an excitation of 800 nm). Exponential fits are used to determine actual scattering properties (see text). b) Percent improvement in fluorescence collection as a function of depth for the optimized vs. “Lens 2 only” configuration.

Fig. 8
Fig. 8

In vivo imaging of Thy1-YFP + P30 mouse cortex. a) Cross-sectional maximum intensity (y-z) projection of a 1000 μm stack containing 500 1024 x 1024 images collected at 2 μm intervals. Scale bar = 100 μm. b) and c) Layer V cell bodies illustrated by way of a maximum intensity z-projection of 50 images from a depth of 750 – 850 μm for an optimized (b) and non-optimized (c) fluorescence collection system. Scale bar = 25 μm. c) and d) 3.5X zoom of a layer V cell body from images (b) and (c), respectively. Scale bar = 10 μm.

Tables (1)

Tables Icon

Table 1 Tissue Phantom Scattering Properties.

Equations (3)

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

F g e n = η σ τ f 2 ( π ( N A ) 2 h c λ ) 2 P 2 e 2 z / l s e x c
F c o l l = ϕ ( z , l s f l u o r , g f l u o r , θ s y s ) F g e n ( z , l s e x c , P , τ , f , ... )
P = P o e z / l s e x c

Metrics