Abstract

We study the effects of primary spherical aberration on the three-dimensional point spread function (PSF) of the two-color (two-photon) excitation (2CE) (2PE) fluorescence microscope with two confocal excitation beams that are separated by an angle θ. The two excitation wavelengths λ1 and λ2 are related to the single-photon excitation wavelength λe by: 1/λe = 1/λ1 + 1/λ2. The general case is considered where both focused beams independently suffer from spherical aberration. For θ = 0, π/2, and π, the resulting deterioration of the PSF structure is evaluated for different values of the spherical aberration coefficients via the Linfoot’s criteria of fidelity, structural content, and correlation quality. The corresponding degradation of the peak 2CE fluorescence intensity is also determined. Our findings are compared with that of the 2PE fluorescence (λ1 = λ2) under the same aberration conditions. We found that the 2CE microscope is more robust against spherical aberration than its 2PE counterpart, with the π/2 configuration providing the clearest advantage. The prospect of aberration correction in the two-beam 2CE microscope is also discussed.

© 2003 Optical Society of America

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References

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    [CrossRef]
  2. M. Lim, C. Saloma, “Confocality condition in two-color excitation microscopy with two focused beams,” Opt. Commun. 207, 111–120 (2002).
    [CrossRef]
  3. M. Cambaliza, C. Saloma, “Advantages of two-color excitation fluorescence microscopy with two confocal excitation beams,” Opt. Commun. 184, 25–35 (2000).
    [CrossRef]
  4. C. Blanca, C. Saloma, “Two-color excitation fluorescence microscopy through highly-scattering media,” Appl. Opt. 70, 2722–2729 (2001).
    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]

2002 (2)

J. Palero, W. Garcia, C. Saloma, “Two-color (two-photon) excitation microscopy with two confocal beams and a Raman shifter,” Opt. Commun. 211, 57–63 (2002).
[CrossRef]

M. Lim, C. Saloma, “Confocality condition in two-color excitation microscopy with two focused beams,” Opt. Commun. 207, 111–120 (2002).
[CrossRef]

2001 (3)

C. Blanca, C. Saloma, “Two-color excitation fluorescence microscopy through highly-scattering media,” Appl. Opt. 70, 2722–2729 (2001).
[CrossRef]

S. Lee, W. Inami, Y. Kawata, “Volume holographic device for spherical aberration correction and parallel data access in three-dimensional memory,” Jap. J. Appl. Phys. 40, 1796–1797 (2001).
[CrossRef]

T. Shimano, M. Umeda, T. Ariyoshi, “Spherical aberration detection in the optical pickups for high-density digital versatile discs,” Jap. J. Appl. Phys. 40, 2292–2295 (2001).
[CrossRef]

2000 (4)

M. Booth, T. Wilson, “Strategies for the compensation of specimen-induced spherical aberration in confocal microscopy of skin,” J. Microsc. 200, 68–74 (2000).
[CrossRef] [PubMed]

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

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

M. Lim, C. Saloma, “Enhancement of low-resolution Raman spectra by simplex projection,” Opt. Commun. 186, 237–243 (2000).
[CrossRef]

1999 (1)

1998 (3)

1997 (2)

P. Török, S. Hewlett, P. Varga, “The role of specimen-induced spherical aberration in confocal microscopy,” J. Microsc. 188, 158–172 (1997).
[CrossRef]

C. Sheppard, P. Torok, “Effects of specimen refractive index on confocal imaging,” J. Microsc. 185, 366–374 (1997).
[CrossRef]

1996 (1)

1990 (1)

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

1985 (1)

Ariyoshi, T.

T. Shimano, M. Umeda, T. Ariyoshi, “Spherical aberration detection in the optical pickups for high-density digital versatile discs,” Jap. J. Appl. Phys. 40, 2292–2295 (2001).
[CrossRef]

Blanca, C.

C. Blanca, C. Saloma, “Two-color excitation fluorescence microscopy through highly-scattering media,” Appl. Opt. 70, 2722–2729 (2001).
[CrossRef]

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

Booth, M.

M. Booth, T. Wilson, “Strategies for the compensation of specimen-induced spherical aberration in confocal microscopy of skin,” J. Microsc. 200, 68–74 (2000).
[CrossRef] [PubMed]

Born, M.

M. Born, E. Wolf, Principles of Optics, 7th ed. (Cambridge University, Cambridge, UK, 1999).

Cambaliza, M.

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

Denk, W.

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

Fales, C.

Garcia, W.

J. Palero, W. Garcia, C. Saloma, “Two-color (two-photon) excitation microscopy with two confocal beams and a Raman shifter,” Opt. Commun. 211, 57–63 (2002).
[CrossRef]

Haylo, N.

Hewlett, S.

P. Török, S. Hewlett, P. Varga, “The role of specimen-induced spherical aberration in confocal microscopy,” J. Microsc. 188, 158–172 (1997).
[CrossRef]

Huck, F.

Inami, W.

S. Lee, W. Inami, Y. Kawata, “Volume holographic device for spherical aberration correction and parallel data access in three-dimensional memory,” Jap. J. Appl. Phys. 40, 1796–1797 (2001).
[CrossRef]

Kawata, Y.

S. Lee, W. Inami, Y. Kawata, “Volume holographic device for spherical aberration correction and parallel data access in three-dimensional memory,” Jap. J. Appl. Phys. 40, 1796–1797 (2001).
[CrossRef]

Lee, S.

S. Lee, W. Inami, Y. Kawata, “Volume holographic device for spherical aberration correction and parallel data access in three-dimensional memory,” Jap. J. Appl. Phys. 40, 1796–1797 (2001).
[CrossRef]

Lim, M.

M. Lim, C. Saloma, “Confocality condition in two-color excitation microscopy with two focused beams,” Opt. Commun. 207, 111–120 (2002).
[CrossRef]

M. Lim, C. Saloma, “Enhancement of low-resolution Raman spectra by simplex projection,” Opt. Commun. 186, 237–243 (2000).
[CrossRef]

Lindek, S.

Nazario, M.

Palero, J.

J. Palero, W. Garcia, C. Saloma, “Two-color (two-photon) excitation microscopy with two confocal beams and a Raman shifter,” Opt. Commun. 211, 57–63 (2002).
[CrossRef]

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 microscope and FISH,” J. Struct. Biol. 131, 56–66 (2000).
[CrossRef] [PubMed]

Saloma, C.

M. Lim, C. Saloma, “Confocality condition in two-color excitation microscopy with two focused beams,” Opt. Commun. 207, 111–120 (2002).
[CrossRef]

J. Palero, W. Garcia, C. Saloma, “Two-color (two-photon) excitation microscopy with two confocal beams and a Raman shifter,” Opt. Commun. 211, 57–63 (2002).
[CrossRef]

C. Blanca, C. Saloma, “Two-color excitation fluorescence microscopy through highly-scattering media,” Appl. Opt. 70, 2722–2729 (2001).
[CrossRef]

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

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

M. Lim, C. Saloma, “Enhancement of low-resolution Raman spectra by simplex projection,” Opt. Commun. 186, 237–243 (2000).
[CrossRef]

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

M. Nazario, C. Saloma, “Signal recovery in sinusoid crossing sampling using the minimum negativity constraint,” Appl. Opt. 37, 2953–2963 (1998).
[CrossRef]

Samms, R.

Sheppard, C.

C. Sheppard, P. Torok, “Effects of specimen refractive index on confocal imaging,” J. Microsc. 185, 366–374 (1997).
[CrossRef]

Shimano, T.

T. Shimano, M. Umeda, T. Ariyoshi, “Spherical aberration detection in the optical pickups for high-density digital versatile discs,” Jap. J. Appl. Phys. 40, 2292–2295 (2001).
[CrossRef]

Stacey, K.

Stelzer, E.

Strickler, J. H.

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

Torok, P.

P. Torok, “Focusing of electromagnetic waves through a dielectric interface by lenses of finite Fresnel number,” J. Opt. Soc. Am. A 15, 3009–3015 (1998).
[CrossRef]

C. Sheppard, P. Torok, “Effects of specimen refractive index on confocal imaging,” J. Microsc. 185, 366–374 (1997).
[CrossRef]

Török, P.

P. Török, S. Hewlett, P. Varga, “The role of specimen-induced spherical aberration in confocal microscopy,” J. Microsc. 188, 158–172 (1997).
[CrossRef]

Umeda, M.

T. Shimano, M. Umeda, T. Ariyoshi, “Spherical aberration detection in the optical pickups for high-density digital versatile discs,” Jap. J. Appl. Phys. 40, 2292–2295 (2001).
[CrossRef]

Varga, P.

P. Török, S. Hewlett, P. Varga, “The role of specimen-induced spherical aberration in confocal microscopy,” J. Microsc. 188, 158–172 (1997).
[CrossRef]

Webb, W.

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

Wilson, T.

M. Booth, T. Wilson, “Strategies for the compensation of specimen-induced spherical aberration in confocal microscopy of skin,” J. Microsc. 200, 68–74 (2000).
[CrossRef] [PubMed]

Wolf, E.

M. Born, E. Wolf, Principles of Optics, 7th ed. (Cambridge University, Cambridge, UK, 1999).

Appl. Opt. (3)

J. Microsc. (3)

M. Booth, T. Wilson, “Strategies for the compensation of specimen-induced spherical aberration in confocal microscopy of skin,” J. Microsc. 200, 68–74 (2000).
[CrossRef] [PubMed]

P. Török, S. Hewlett, P. Varga, “The role of specimen-induced spherical aberration in confocal microscopy,” J. Microsc. 188, 158–172 (1997).
[CrossRef]

C. Sheppard, P. Torok, “Effects of specimen refractive index on confocal imaging,” J. Microsc. 185, 366–374 (1997).
[CrossRef]

J. Opt. Soc. Am. A (3)

J. Struct. Biol. (1)

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

Jap. J. Appl. Phys. (2)

S. Lee, W. Inami, Y. Kawata, “Volume holographic device for spherical aberration correction and parallel data access in three-dimensional memory,” Jap. J. Appl. Phys. 40, 1796–1797 (2001).
[CrossRef]

T. Shimano, M. Umeda, T. Ariyoshi, “Spherical aberration detection in the optical pickups for high-density digital versatile discs,” Jap. J. Appl. Phys. 40, 2292–2295 (2001).
[CrossRef]

Opt. Commun. (4)

J. Palero, W. Garcia, C. Saloma, “Two-color (two-photon) excitation microscopy with two confocal beams and a Raman shifter,” Opt. Commun. 211, 57–63 (2002).
[CrossRef]

M. Lim, C. Saloma, “Confocality condition in two-color excitation microscopy with two focused beams,” Opt. Commun. 207, 111–120 (2002).
[CrossRef]

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

M. Lim, C. Saloma, “Enhancement of low-resolution Raman spectra by simplex projection,” Opt. Commun. 186, 237–243 (2000).
[CrossRef]

Opt. Lett. (1)

Science (1)

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

Other (1)

M. Born, E. Wolf, Principles of Optics, 7th ed. (Cambridge University, Cambridge, UK, 1999).

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Figures (6)

Fig. 1
Fig. 1

Scanning microscope with two confocal excitation beams oriented at an angle θ with respect to each other. At an observation point P, the field distribution due to L1 is U 1(P; λ1; Φ1) while that due to L2 is U 2(P; λ2; θ; Φ2).

Fig. 2
Fig. 2

Contour plots (xz plane) of aberration-free PSF (ϕ1 = 0, ϕ2 = 0). 2PE (left-hand side column) and 2CE (right-hand side column) at θ = 0 [(a), (d)], π/2 [(b), (e)], and π [(c), (f)]. The common geometrical focus of the two confocal excitation beams is at P(x = 0, y = 0, z = 0). Parameters: λ1 = 750 nm, λ2 = 656 nm, λ e = 350 nm = λ f = λ2p /2, and NA = 0.5.

Fig. 3
Fig. 3

Contour plots (xz plane) of aberrated PSF (ϕ1 = 16, ϕ2 = 16, primary spherical aberration). 2PE (left-hand side column) and 2CE (right-hand side column) at θ = 0 [(a), (d)], π/2 [(b), (e)], and π [(c), (f)]. Parameters: λ1 = 750 nm, λ2 = 656 nm, λ e = 350 nm = λ f = λ2p /2, and NA = 0.5.

Fig. 4
Fig. 4

Dependence (contour plots) of 2PE (left-hand side column) and 2CE (right-hand side column) peak fluorescence intensity with ϕ1 and ϕ2 at θ = 0 [(a), (d)], π/2 [(b), (e)], and π [(c), (f)]. Parameters used: ϕ = 100 Φ/λρ4, λ1 = 750 nm, λ2 = 656 nm, λ e = 350 nm = λ f = λ2p /2 and NA = 0.5.

Fig. 5
Fig. 5

Dependence (contour plots) of 2PE (left-hand side column) and 2CE (right-hand side column) fidelity F with ϕ1 and ϕ2 at θ = 0 [(a), (d)], π/2 [(b), (e)], and π [(c), (f)]. Parameters used: ϕ = 100 Φ/λρ4, λ1 = 750 nm, λ2 = 656 nm, λ e = 350 nm = λ f = λ2p /2 and NA = 0.5.

Fig. 6
Fig. 6

Dependence (contour plots) of 2PE (left-hand side column) and 2CE (right-hand side column) correlation quality Q with ϕ1 and ϕ2 at θ = 0 [(a), (d)], π/2 [(b), (e)], and π [(c), (f)]. Parameters used: ϕ = 100 Φ/λρ4, λ1 = 750 nm, λ2 = 656 nm, λ e = 350 nm = λ f = λ2p /2 and NA = 0.5.

Equations (1)

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Uu, v, ψ= -iλAa2R2expiR/a2u 0102πexpikΦ-vρ cosδ-ψ- 12 uρ2ρdρdδ,

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