Abstract

We study the performance of two-color excitation (2CE) fluorescence microscopy [Opt. Lett. 24, 1505 (1999)] in turbid media of different densities and anisotropy. Excitation is achieved with two confocal excitation beams of wavelengths λ1 and λ2, which are separated by an angular displacement θ, where λ1 ≠ λ2, 1/λe = 1/λ1 + 1/λ2, and λe is the single-photon excitation wavelength of the sample. 2CE fluorescence is generated only in regions of the sample where the two excitation beams overlap. The 2CE fluorescence intensity is proportional to the product of the two excitation intensities and could be detected with a large-area photodetector. The requirement of spatiotemporal simultaneity for the two excitation beams makes 2CE fluorescence imaging a promising tool for observing microscopic objects in a highly scattering medium. Optical scattering asymmetrically broadens the excitation point-spread function and toward the side of the focusing lens that leads to the contrast deterioration of the fluorescence image in single- or two-photon (λ1 = λ2) excitation. Image degradation is caused by the decrease in the excitation energy density at the geometrical focus and by the increase in background fluorescence from the out-of-focus planes. In a beam configuration with θ ≠ 0, 2CE fluorescence imaging is robust against the deleterious effects of scattering on the excitation-beam distribution. Scattering only decreases the available energy density at the geometrical focus and does not increase the background noise. For both isotropic and anisotropic scattering media the performance of 2CE imaging is studied with a Monte Carlo simulation for θ = 0, π/2, and π, and at different h/d s values where h is the scattering depth and d s is the mean-free path of the scattering medium.

© 2001 Optical Society of America

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  1. M. Nagorni, S. Hell, “4Pi-confocal microscopy provides three-dimensional images of the microtubule network with 100- to 150-nm resolution,” J. Struct. Biol. 123, 236–247 (1998).
    [CrossRef]
  2. W. Denk, J. H. Strickler, W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
    [CrossRef] [PubMed]
  3. V. Daria, C. Blanca, O. Nakamura, S. Kawata, C. Saloma, “Image contrast enhancement for two-photon fluorescence microscopy in a turbid medium,” Appl. Opt. 37, 7960–7967 (1998).
    [CrossRef]
  4. S. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, J. Lakowicz, “Three-photon excitation in fluorescence microscopy,” J. Biomed. Opt. 1, 71–74 (1996).
    [CrossRef] [PubMed]
  5. S. Lindek, C. Cremer, E. Stelzer, “Confocal theta fluorescence microscopy with annular apertures,” Appl. Opt. 35, 126–130 (1996).
    [CrossRef] [PubMed]
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    [CrossRef]
  7. S. Hell, M. Nagorni, “4Pi confocal microscopy with alternate interference,” Opt. Lett. 23, 1567–1569 (1998).
    [CrossRef]
  8. J. Lakowicz, I. Gryczynski, H. Malak, Z. Gryczynski, “Fluorescence spectral properties of 2,5-Diphenyl-1,3,4-oxadiazole with two-color two-photon excitation,” J. Phys. Chem. 100, 19406–19411 (1996).
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    [CrossRef]
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    [CrossRef]
  12. C. Palmes-Saloma, C. Saloma, “Long-depth imaging of specific gene expressions in wholemount mouse embryos with single photon excitation confocal fluorescence microscopy and FISH,” J. Struct. Biol. 131, 56–66 (2000).
    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
  17. J. W. Goodman, Introduction to Fourier Optics (McGraw-Hill, New York, 1968).
  18. R. G. Newton, Scattering Theory of Waves and Particles (McGraw-Hill, New York, 1966).
  19. J. Ying, F. Liu, R. Alfano, “Spatial distribution of two-photon-excited fluorescence in scattering media,” Appl. Opt. 18, 224–230 (1999).
    [CrossRef]
  20. H. C. van de Hulst, Multiple Scattering Light Tables, Formulas and Applications (Academic, New York, 1980).

2000 (2)

M. O. Cambaliza, C. Saloma, “Advantages of two-color excitation fluorescence microscopy with two confocal excitation beams,” Opt. Commun. 184, 25–35 (2000).
[CrossRef]

C. Palmes-Saloma, C. Saloma, “Long-depth imaging of specific gene expressions in wholemount mouse embryos with single photon excitation confocal fluorescence microscopy and FISH,” J. Struct. Biol. 131, 56–66 (2000).
[CrossRef] [PubMed]

1999 (4)

L. Mandel, “Quantum effects in one-photon and two-photon interference,” Rev. Mod. Phys. 71, S274–S282 (1999).
[CrossRef]

J. Ying, F. Liu, R. Alfano, “Spatial distribution of two-photon-excited fluorescence in scattering media,” Appl. Opt. 18, 224–230 (1999).
[CrossRef]

S. Lindek, E. Stelzer, “Resolution improvement by nonconfocal theta microscopy,” Opt. Lett. 24, 1505–1507 (1999).
[CrossRef]

C. Blanca, C. Saloma, “Efficient analysis of temporal broadening of a pulse focused Gaussian beam in scattering media,” Appl. Opt. 38, 5433–5437 (1999).
[CrossRef]

1998 (4)

1996 (4)

S. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, J. Lakowicz, “Three-photon excitation in fluorescence microscopy,” J. Biomed. Opt. 1, 71–74 (1996).
[CrossRef] [PubMed]

J. Lakowicz, I. Gryczynski, H. Malak, Z. Gryczynski, “Fluorescence spectral properties of 2,5-Diphenyl-1,3,4-oxadiazole with two-color two-photon excitation,” J. Phys. Chem. 100, 19406–19411 (1996).
[CrossRef]

S. Lindek, E. Stelzer, “Optical transfer functions for confocal-theta fluorescence microscopy,” J. Opt. Soc. Am. A 13, 479–482 (1996).
[CrossRef]

S. Lindek, C. Cremer, E. Stelzer, “Confocal theta fluorescence microscopy with annular apertures,” Appl. Opt. 35, 126–130 (1996).
[CrossRef] [PubMed]

1990 (2)

W. Cheong, S. Prahl, A. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[CrossRef]

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

1941 (1)

L. Henyey, J. Greenstein, “Diffuse radiation in the galaxy,” Astrophys. J. 93, 70–83 (1941).
[CrossRef]

Alfano, R.

J. Ying, F. Liu, R. Alfano, “Spatial distribution of two-photon-excited fluorescence in scattering media,” Appl. Opt. 18, 224–230 (1999).
[CrossRef]

Bahlmann, K.

S. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, J. Lakowicz, “Three-photon excitation in fluorescence microscopy,” J. Biomed. Opt. 1, 71–74 (1996).
[CrossRef] [PubMed]

Blanca, C.

Cambaliza, M. O.

M. O. Cambaliza, C. Saloma, “Advantages of two-color excitation fluorescence microscopy with two confocal excitation beams,” Opt. Commun. 184, 25–35 (2000).
[CrossRef]

Cheong, W.

W. Cheong, S. Prahl, A. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[CrossRef]

Cremer, C.

Daria, V.

Denk, W.

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

Goodman, J. W.

J. W. Goodman, Introduction to Fourier Optics (McGraw-Hill, New York, 1968).

Greenstein, J.

L. Henyey, J. Greenstein, “Diffuse radiation in the galaxy,” Astrophys. J. 93, 70–83 (1941).
[CrossRef]

Gryczynski, I.

S. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, J. Lakowicz, “Three-photon excitation in fluorescence microscopy,” J. Biomed. Opt. 1, 71–74 (1996).
[CrossRef] [PubMed]

J. Lakowicz, I. Gryczynski, H. Malak, Z. Gryczynski, “Fluorescence spectral properties of 2,5-Diphenyl-1,3,4-oxadiazole with two-color two-photon excitation,” J. Phys. Chem. 100, 19406–19411 (1996).
[CrossRef]

Gryczynski, Z.

J. Lakowicz, I. Gryczynski, H. Malak, Z. Gryczynski, “Fluorescence spectral properties of 2,5-Diphenyl-1,3,4-oxadiazole with two-color two-photon excitation,” J. Phys. Chem. 100, 19406–19411 (1996).
[CrossRef]

Hell, S.

S. Hell, M. Nagorni, “4Pi confocal microscopy with alternate interference,” Opt. Lett. 23, 1567–1569 (1998).
[CrossRef]

M. Nagorni, S. Hell, “4Pi-confocal microscopy provides three-dimensional images of the microtubule network with 100- to 150-nm resolution,” J. Struct. Biol. 123, 236–247 (1998).
[CrossRef]

S. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, J. Lakowicz, “Three-photon excitation in fluorescence microscopy,” J. Biomed. Opt. 1, 71–74 (1996).
[CrossRef] [PubMed]

Henyey, L.

L. Henyey, J. Greenstein, “Diffuse radiation in the galaxy,” Astrophys. J. 93, 70–83 (1941).
[CrossRef]

Kawata, S.

Lakowicz, J.

S. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, J. Lakowicz, “Three-photon excitation in fluorescence microscopy,” J. Biomed. Opt. 1, 71–74 (1996).
[CrossRef] [PubMed]

J. Lakowicz, I. Gryczynski, H. Malak, Z. Gryczynski, “Fluorescence spectral properties of 2,5-Diphenyl-1,3,4-oxadiazole with two-color two-photon excitation,” J. Phys. Chem. 100, 19406–19411 (1996).
[CrossRef]

Lindek, S.

Liu, F.

J. Ying, F. Liu, R. Alfano, “Spatial distribution of two-photon-excited fluorescence in scattering media,” Appl. Opt. 18, 224–230 (1999).
[CrossRef]

Malak, H.

J. Lakowicz, I. Gryczynski, H. Malak, Z. Gryczynski, “Fluorescence spectral properties of 2,5-Diphenyl-1,3,4-oxadiazole with two-color two-photon excitation,” J. Phys. Chem. 100, 19406–19411 (1996).
[CrossRef]

S. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, J. Lakowicz, “Three-photon excitation in fluorescence microscopy,” J. Biomed. Opt. 1, 71–74 (1996).
[CrossRef] [PubMed]

Mandel, L.

L. Mandel, “Quantum effects in one-photon and two-photon interference,” Rev. Mod. Phys. 71, S274–S282 (1999).
[CrossRef]

Nagorni, M.

M. Nagorni, S. Hell, “4Pi-confocal microscopy provides three-dimensional images of the microtubule network with 100- to 150-nm resolution,” J. Struct. Biol. 123, 236–247 (1998).
[CrossRef]

S. Hell, M. Nagorni, “4Pi confocal microscopy with alternate interference,” Opt. Lett. 23, 1567–1569 (1998).
[CrossRef]

Nakamura, O.

Newton, R. G.

R. G. Newton, Scattering Theory of Waves and Particles (McGraw-Hill, New York, 1966).

Palmes-Saloma, C.

C. Palmes-Saloma, C. Saloma, “Long-depth imaging of specific gene expressions in wholemount mouse embryos with single photon excitation confocal fluorescence microscopy and FISH,” J. Struct. Biol. 131, 56–66 (2000).
[CrossRef] [PubMed]

Prahl, S.

W. Cheong, S. Prahl, A. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[CrossRef]

Saloma, C.

C. Palmes-Saloma, C. Saloma, “Long-depth imaging of specific gene expressions in wholemount mouse embryos with single photon excitation confocal fluorescence microscopy and FISH,” J. Struct. Biol. 131, 56–66 (2000).
[CrossRef] [PubMed]

M. O. Cambaliza, C. Saloma, “Advantages of two-color excitation fluorescence microscopy with two confocal excitation beams,” Opt. Commun. 184, 25–35 (2000).
[CrossRef]

C. Blanca, C. Saloma, “Efficient analysis of temporal broadening of a pulse focused Gaussian beam in scattering media,” Appl. Opt. 38, 5433–5437 (1999).
[CrossRef]

V. Daria, C. Blanca, O. Nakamura, S. Kawata, C. Saloma, “Image contrast enhancement for two-photon fluorescence microscopy in a turbid medium,” Appl. Opt. 37, 7960–7967 (1998).
[CrossRef]

C. Blanca, C. Saloma, “Monte Carlo analysis of two-photon fluorescence imaging through a scattering medium,” Appl. Opt. 37, 8092–8102 (1998).
[CrossRef]

Schrader, M.

S. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, J. Lakowicz, “Three-photon excitation in fluorescence microscopy,” J. Biomed. Opt. 1, 71–74 (1996).
[CrossRef] [PubMed]

Soini, A.

S. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, J. Lakowicz, “Three-photon excitation in fluorescence microscopy,” J. Biomed. Opt. 1, 71–74 (1996).
[CrossRef] [PubMed]

Stelzer, E.

Strickler, J. H.

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

van de Hulst, H. C.

H. C. van de Hulst, Multiple Scattering Light Tables, Formulas and Applications (Academic, New York, 1980).

Webb, W.

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

Welch, A.

W. Cheong, S. Prahl, A. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[CrossRef]

Ying, J.

J. Ying, F. Liu, R. Alfano, “Spatial distribution of two-photon-excited fluorescence in scattering media,” Appl. Opt. 18, 224–230 (1999).
[CrossRef]

Appl. Opt. (5)

Astrophys. J. (1)

L. Henyey, J. Greenstein, “Diffuse radiation in the galaxy,” Astrophys. J. 93, 70–83 (1941).
[CrossRef]

IEEE J. Quantum Electron. (1)

W. Cheong, S. Prahl, A. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[CrossRef]

J. Biomed. Opt. (1)

S. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, J. Lakowicz, “Three-photon excitation in fluorescence microscopy,” J. Biomed. Opt. 1, 71–74 (1996).
[CrossRef] [PubMed]

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

J. Phys. Chem. (1)

J. Lakowicz, I. Gryczynski, H. Malak, Z. Gryczynski, “Fluorescence spectral properties of 2,5-Diphenyl-1,3,4-oxadiazole with two-color two-photon excitation,” J. Phys. Chem. 100, 19406–19411 (1996).
[CrossRef]

J. Struct. Biol. (2)

M. Nagorni, S. Hell, “4Pi-confocal microscopy provides three-dimensional images of the microtubule network with 100- to 150-nm resolution,” J. Struct. Biol. 123, 236–247 (1998).
[CrossRef]

C. Palmes-Saloma, C. Saloma, “Long-depth imaging of specific gene expressions in wholemount mouse embryos with single photon excitation confocal fluorescence microscopy and FISH,” J. Struct. Biol. 131, 56–66 (2000).
[CrossRef] [PubMed]

Opt. Commun. (1)

M. O. Cambaliza, C. Saloma, “Advantages of two-color excitation fluorescence microscopy with two confocal excitation beams,” Opt. Commun. 184, 25–35 (2000).
[CrossRef]

Opt. Lett. (2)

Rev. Mod. Phys. (1)

L. Mandel, “Quantum effects in one-photon and two-photon interference,” Rev. Mod. Phys. 71, S274–S282 (1999).
[CrossRef]

Science (1)

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

Other (3)

J. W. Goodman, Introduction to Fourier Optics (McGraw-Hill, New York, 1968).

R. G. Newton, Scattering Theory of Waves and Particles (McGraw-Hill, New York, 1966).

H. C. van de Hulst, Multiple Scattering Light Tables, Formulas and Applications (Academic, New York, 1980).

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Figures (9)

Fig. 1
Fig. 1

Configuration of 2CE with two confocal focused excitation beams separated by an angle θ. Fluorescence is generated only in regions where the two excitation beams I 1(P; λ1, θ1) and I 2(P; λ2, θ2) are simultaneously present in space and in time. The generated fluorescence signal is detected by a large-area photodetector (not shown). The focusing objective lenses (L1, L2) are identical where λ e is the single-photon excitation wavelength and P is the observation point.

Fig. 2
Fig. 2

Anisotropic medium (g = 0.9). Time-integrated (normalized) intensity distribution of a focused excitation beam propagating in media of different densities. Illumination is along the vertical z axis, and the dashed lines outline the geometrical boundaries of the focused beam. The beam propagates from the top. Pertinent parameters: N.A. = 0.7, N e = 107, λ2P = 700 nm, and h = 1 mm. The geometrical focus is located at the center of the frame (size, 0.5 mm × 0.5 mm).

Fig. 3
Fig. 3

Isotropic medium (g = 0.0001). Time-integrated intensity distribution of a focused beam propagating in media with different densities. Pertinent parameters: N.A. = 0.7, N e = 107, λ2P = 700 nm, and h = 1 mm. The geometrical focus is located at the center of the frame (size, 0.5 mm × 0.5 mm).

Fig. 4
Fig. 4

Normalized axial excitation intensity profiles I(z, λ2P ) at different R values for g = 0.9 (bold curves) and g = 0.0001 (thin curves). The reference (unscattered) excitation profile (d s = 106) is plotted in dotted curves. The negative z axis consists of coordinates on the side of the sample interface. Pertinent parameters: N.A. = 0.7, N e = 107, λ2P = 700 nm, and h = 1 mm.

Fig. 5
Fig. 5

Normalized 2PE fluorescence profiles at different R values for g = 0.9 (bold curves) and g = 0.0001 (thin curves). The reference (unscattered) excitation profile (d s = 106) is plotted in dotted curves. Pertinent parameters: N.A. = 0.7, N e = 107, λ2P = 700 nm, and h = 1 mm.

Fig. 6
Fig. 6

Anisotropic medium (g = 0.9). Normalized 2PE and 2CE fluorescence profiles obtained under (θ = π)-excitation configuration (4pi). The reference (unscattered) excitation profile (d s = 106) is plotted in dotted curves. For 2CE: λ1 = 750 nm (crosses) and λ2 = 656 nm (circles). Other pertinent parameters: N.A. = 0.7, N e = 107, λ2P = 700 nm, and h = 1 mm.

Fig. 7
Fig. 7

Isotropic medium (g = 0.0001). Normalized 2PE and 2CE fluorescence profiles obtained under (θ = π)-excitation configuration (4pi). The reference (unscattered) excitation profile (d s = 106) is plotted in dotted curves. For 2CE: λ1 = 750 nm (crosses) and λ2 = 656 nm (circles). Other pertinent parameters: N.A. = 0.7, N e = 107, λ2P = 700 nm, and h = 1 mm.

Fig. 8
Fig. 8

Anisotropic medium (g = 0.9). Normalized 2PE (thin curves) and 2CE (bold curves) fluorescence profiles under three different θ excitation configurations (R = 8): θ = 0, π/2, and π. Pertinent parameters: N.A. = 0.7, N e = 107, λ2P = 700 nm, λ1 = 750 nm, λ2 = 656 nm, and h = 1 mm.

Fig. 9
Fig. 9

Isotropic medium (g = 0.0001). Normalized 2PE (thin curves) and 2CE (bold curves) fluorescence profiles under three different θ-excitation configurations (R = 8): θ = 0, π/2, and π. Pertinent parameters: N.A. = 0.7, N e = 107, λ2P = 700 nm, λ1 = 750 nm, λ2 = 656 nm, and h = 1 mm.

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