Abstract

A numerical model was developed to simulate the effects of tissue optical properties, objective numerical aperture (N.A.), and instrument performance on two-photon-excited fluorescence imaging of turbid samples. Model data are compared with measurements of fluorescent microspheres in a tissuelike scattering phantom. Our results show that the measured two-photon-excited signal decays exponentially with increasing focal depth. The overall decay constant is a function of absorption and scattering parameters at both excitation and emission wavelengths. The generation of two-photon fluorescence is shown to be independent of the scattering anisotropy, g, except for g > 0.95. The N.A. for which the maximum signal is collected varies with depth, although this effect is not seen until the focal plane is greater than two scattering mean free paths into the sample. Overall, measurements and model results indicate that resolution in two-photon microscopy is dependent solely on the ability to deliver sufficient ballistic photon density to the focal volume. As a result we show that lateral resolution in two-photon microscopy is largely unaffected by tissue optical properties in the range typically encountered in soft tissues, although the maximum imaging depth is strongly dependent on absorption and scattering coefficients, scattering anisotropy, and objective N.A..

© 2000 Optical Society of America

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References

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  1. W. Denk, J. Strickler, W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 78–76 (1990).
    [CrossRef]
  2. K. H. Kim, C. Buehler, C.-Y. Dong, B. R. Masters, P. T. C. So, “Tissue imaging using two-photon video rate microscopy,” in Optical Diagnostics of Living Cells II, D. L. Farkas, R. C. Leif, B. J. Tromberg, eds., Proc. SPIE3604, 60–66 (1999).
    [CrossRef]
  3. D. Kleinfeld, P. Mitra, F. Helmchen, W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. USA 95, 15741–15746 (1998).
    [CrossRef] [PubMed]
  4. J. Schmitt, A. Knuttel, M. Yadlowski, “Confocal microscopy in turbid media,” J. Opt. Soc. Am. A 11, 2226–2235 (1994).
    [CrossRef]
  5. X. Gan, S. Schilders, M. Gu, “Image formation in turbid media under a microscope,” J. Opt. Soc. Am. A 15, 2052–2058 (1998).
    [CrossRef]
  6. A. Dunn, C. Smithpeter, R. Richards-Kortum, A. J. Welch, “Sources of contrast in confocal reflectance imaging,” Appl. Opt. 35, 3441–3446 (1996).
    [CrossRef] [PubMed]
  7. D. Smithies, T. Lindmo, Z. Chen, J. Nelson, T. Milner, “Signal attenuation and localization in optical coherence tomography studied by Monte Carlo simulation,” Phys. Med. Biol. 43, 3025–3044 (1998).
    [CrossRef] [PubMed]
  8. X. Gan, M. Gu, “Effective point-spread function for fast image modeling and processing in microscopic imaging through turbid media,” Opt. Lett. 24, 741–743 (1999).
    [CrossRef]
  9. C. Xu, 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, 481–491 (1996).
    [CrossRef]
  10. J. Fishkin, O. Coquoz, E. Anderson, M. Brenner, B. Tromberg, “Frequency-domain photon migration measurements of normal and malignant tissue optical properties in a human subject,” Appl. Opt. 36, 10–20 (1997).
    [CrossRef] [PubMed]
  11. C. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).
  12. S. Jacques, C. Alter, S. Prahl, “Angular dependence of HeNe laser light scattering by human dermis,” Lasers Life Sci. 1, 309–333 (1987).
  13. A. Dunn, R. Richards-Kortum, “Three-dimensional computation of light scattering from cells,” IEEE J. Sel. Top. Quantum Electron. 2, 898–905 (1997).
    [CrossRef]
  14. H. van Staveren, C. Moes, J. van Marle, S. Prahl, M. van Gemert, “Light scattering in Intralipid-10% in the wavelength range of 400–1100 nm,” Appl. Opt. 30, 4507–4514 (1991).
    [CrossRef] [PubMed]
  15. C. L. Smithpeter, A. K. Dunn, A. J. Welch, R. Richards-Kortum, “Penetration depth limits of in vivo confocal reflectance imaging,” Appl. Opt. 37, 2749–2754 (1998).
    [CrossRef]
  16. V. Centonze, J. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75, 2015–2024 (1998).
    [CrossRef] [PubMed]
  17. H. Kume, ed., Hamamatsu Photonics, in Photomultiplier Tube: Principle to Application, (Hamamatsu Photonics, Bridgewater, N.J., 1994).
  18. R. Hornung, T. Pham, K. Keefe, J. Fishkin, M. Berns, Y. Tadir, B. Tromberg, “Quantitative near infrared spectroscopy of cervical dysplasia in vivo,” Hum. Reprod. 14, 2908–2916 (1999).
    [CrossRef] [PubMed]
  19. T. Farrel, B. Wilson, M. Patterson, M. Olivio, “Comparison of the in vivo photodynamic threshold dose for photofrin, mono- and tetrasulfonated aluminum phthalocyanine using a rat liver model,” Photochem. Photobiol. 68, 394–399 (1998).
    [CrossRef]
  20. D. Oh, R. Stanley, M. Lin, W. Hoeffler, S. Boxer, M. Berns, E. Bauer, “Two-photon excitation of 4′-Hydroxylmethyl-4,5′,8-Trimethylpsoralen,” Photochem. Photobiol. 65, 91–95 (1997).
    [CrossRef] [PubMed]

1999 (2)

X. Gan, M. Gu, “Effective point-spread function for fast image modeling and processing in microscopic imaging through turbid media,” Opt. Lett. 24, 741–743 (1999).
[CrossRef]

R. Hornung, T. Pham, K. Keefe, J. Fishkin, M. Berns, Y. Tadir, B. Tromberg, “Quantitative near infrared spectroscopy of cervical dysplasia in vivo,” Hum. Reprod. 14, 2908–2916 (1999).
[CrossRef] [PubMed]

1998 (6)

T. Farrel, B. Wilson, M. Patterson, M. Olivio, “Comparison of the in vivo photodynamic threshold dose for photofrin, mono- and tetrasulfonated aluminum phthalocyanine using a rat liver model,” Photochem. Photobiol. 68, 394–399 (1998).
[CrossRef]

C. L. Smithpeter, A. K. Dunn, A. J. Welch, R. Richards-Kortum, “Penetration depth limits of in vivo confocal reflectance imaging,” Appl. Opt. 37, 2749–2754 (1998).
[CrossRef]

V. Centonze, J. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75, 2015–2024 (1998).
[CrossRef] [PubMed]

D. Smithies, T. Lindmo, Z. Chen, J. Nelson, T. Milner, “Signal attenuation and localization in optical coherence tomography studied by Monte Carlo simulation,” Phys. Med. Biol. 43, 3025–3044 (1998).
[CrossRef] [PubMed]

D. Kleinfeld, P. Mitra, F. Helmchen, W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. USA 95, 15741–15746 (1998).
[CrossRef] [PubMed]

X. Gan, S. Schilders, M. Gu, “Image formation in turbid media under a microscope,” J. Opt. Soc. Am. A 15, 2052–2058 (1998).
[CrossRef]

1997 (3)

D. Oh, R. Stanley, M. Lin, W. Hoeffler, S. Boxer, M. Berns, E. Bauer, “Two-photon excitation of 4′-Hydroxylmethyl-4,5′,8-Trimethylpsoralen,” Photochem. Photobiol. 65, 91–95 (1997).
[CrossRef] [PubMed]

J. Fishkin, O. Coquoz, E. Anderson, M. Brenner, B. Tromberg, “Frequency-domain photon migration measurements of normal and malignant tissue optical properties in a human subject,” Appl. Opt. 36, 10–20 (1997).
[CrossRef] [PubMed]

A. Dunn, R. Richards-Kortum, “Three-dimensional computation of light scattering from cells,” IEEE J. Sel. Top. Quantum Electron. 2, 898–905 (1997).
[CrossRef]

1996 (2)

1994 (1)

1991 (1)

1990 (2)

C. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).

W. Denk, J. Strickler, W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 78–76 (1990).
[CrossRef]

1987 (1)

S. Jacques, C. Alter, S. Prahl, “Angular dependence of HeNe laser light scattering by human dermis,” Lasers Life Sci. 1, 309–333 (1987).

Alter, C.

S. Jacques, C. Alter, S. Prahl, “Angular dependence of HeNe laser light scattering by human dermis,” Lasers Life Sci. 1, 309–333 (1987).

Anderson, E.

Bauer, E.

D. Oh, R. Stanley, M. Lin, W. Hoeffler, S. Boxer, M. Berns, E. Bauer, “Two-photon excitation of 4′-Hydroxylmethyl-4,5′,8-Trimethylpsoralen,” Photochem. Photobiol. 65, 91–95 (1997).
[CrossRef] [PubMed]

Berns, M.

R. Hornung, T. Pham, K. Keefe, J. Fishkin, M. Berns, Y. Tadir, B. Tromberg, “Quantitative near infrared spectroscopy of cervical dysplasia in vivo,” Hum. Reprod. 14, 2908–2916 (1999).
[CrossRef] [PubMed]

D. Oh, R. Stanley, M. Lin, W. Hoeffler, S. Boxer, M. Berns, E. Bauer, “Two-photon excitation of 4′-Hydroxylmethyl-4,5′,8-Trimethylpsoralen,” Photochem. Photobiol. 65, 91–95 (1997).
[CrossRef] [PubMed]

Boxer, S.

D. Oh, R. Stanley, M. Lin, W. Hoeffler, S. Boxer, M. Berns, E. Bauer, “Two-photon excitation of 4′-Hydroxylmethyl-4,5′,8-Trimethylpsoralen,” Photochem. Photobiol. 65, 91–95 (1997).
[CrossRef] [PubMed]

Brenner, M.

Buehler, C.

K. H. Kim, C. Buehler, C.-Y. Dong, B. R. Masters, P. T. C. So, “Tissue imaging using two-photon video rate microscopy,” in Optical Diagnostics of Living Cells II, D. L. Farkas, R. C. Leif, B. J. Tromberg, eds., Proc. SPIE3604, 60–66 (1999).
[CrossRef]

Centonze, V.

V. Centonze, J. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75, 2015–2024 (1998).
[CrossRef] [PubMed]

Chen, Z.

D. Smithies, T. Lindmo, Z. Chen, J. Nelson, T. Milner, “Signal attenuation and localization in optical coherence tomography studied by Monte Carlo simulation,” Phys. Med. Biol. 43, 3025–3044 (1998).
[CrossRef] [PubMed]

Coquoz, O.

Denk, W.

D. Kleinfeld, P. Mitra, F. Helmchen, W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. USA 95, 15741–15746 (1998).
[CrossRef] [PubMed]

W. Denk, J. Strickler, W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 78–76 (1990).
[CrossRef]

Dong, C.-Y.

K. H. Kim, C. Buehler, C.-Y. Dong, B. R. Masters, P. T. C. So, “Tissue imaging using two-photon video rate microscopy,” in Optical Diagnostics of Living Cells II, D. L. Farkas, R. C. Leif, B. J. Tromberg, eds., Proc. SPIE3604, 60–66 (1999).
[CrossRef]

Dunn, A.

A. Dunn, R. Richards-Kortum, “Three-dimensional computation of light scattering from cells,” IEEE J. Sel. Top. Quantum Electron. 2, 898–905 (1997).
[CrossRef]

A. Dunn, C. Smithpeter, R. Richards-Kortum, A. J. Welch, “Sources of contrast in confocal reflectance imaging,” Appl. Opt. 35, 3441–3446 (1996).
[CrossRef] [PubMed]

Dunn, A. K.

Farrel, T.

T. Farrel, B. Wilson, M. Patterson, M. Olivio, “Comparison of the in vivo photodynamic threshold dose for photofrin, mono- and tetrasulfonated aluminum phthalocyanine using a rat liver model,” Photochem. Photobiol. 68, 394–399 (1998).
[CrossRef]

Fishkin, J.

R. Hornung, T. Pham, K. Keefe, J. Fishkin, M. Berns, Y. Tadir, B. Tromberg, “Quantitative near infrared spectroscopy of cervical dysplasia in vivo,” Hum. Reprod. 14, 2908–2916 (1999).
[CrossRef] [PubMed]

J. Fishkin, O. Coquoz, E. Anderson, M. Brenner, B. Tromberg, “Frequency-domain photon migration measurements of normal and malignant tissue optical properties in a human subject,” Appl. Opt. 36, 10–20 (1997).
[CrossRef] [PubMed]

Gan, X.

Gu, M.

Helmchen, F.

D. Kleinfeld, P. Mitra, F. Helmchen, W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. USA 95, 15741–15746 (1998).
[CrossRef] [PubMed]

Hoeffler, W.

D. Oh, R. Stanley, M. Lin, W. Hoeffler, S. Boxer, M. Berns, E. Bauer, “Two-photon excitation of 4′-Hydroxylmethyl-4,5′,8-Trimethylpsoralen,” Photochem. Photobiol. 65, 91–95 (1997).
[CrossRef] [PubMed]

Hornung, R.

R. Hornung, T. Pham, K. Keefe, J. Fishkin, M. Berns, Y. Tadir, B. Tromberg, “Quantitative near infrared spectroscopy of cervical dysplasia in vivo,” Hum. Reprod. 14, 2908–2916 (1999).
[CrossRef] [PubMed]

Jacques, S.

S. Jacques, C. Alter, S. Prahl, “Angular dependence of HeNe laser light scattering by human dermis,” Lasers Life Sci. 1, 309–333 (1987).

Keefe, K.

R. Hornung, T. Pham, K. Keefe, J. Fishkin, M. Berns, Y. Tadir, B. Tromberg, “Quantitative near infrared spectroscopy of cervical dysplasia in vivo,” Hum. Reprod. 14, 2908–2916 (1999).
[CrossRef] [PubMed]

Kim, K. H.

K. H. Kim, C. Buehler, C.-Y. Dong, B. R. Masters, P. T. C. So, “Tissue imaging using two-photon video rate microscopy,” in Optical Diagnostics of Living Cells II, D. L. Farkas, R. C. Leif, B. J. Tromberg, eds., Proc. SPIE3604, 60–66 (1999).
[CrossRef]

Kleinfeld, D.

D. Kleinfeld, P. Mitra, F. Helmchen, W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. USA 95, 15741–15746 (1998).
[CrossRef] [PubMed]

Knuttel, A.

Lin, M.

D. Oh, R. Stanley, M. Lin, W. Hoeffler, S. Boxer, M. Berns, E. Bauer, “Two-photon excitation of 4′-Hydroxylmethyl-4,5′,8-Trimethylpsoralen,” Photochem. Photobiol. 65, 91–95 (1997).
[CrossRef] [PubMed]

Lindmo, T.

D. Smithies, T. Lindmo, Z. Chen, J. Nelson, T. Milner, “Signal attenuation and localization in optical coherence tomography studied by Monte Carlo simulation,” Phys. Med. Biol. 43, 3025–3044 (1998).
[CrossRef] [PubMed]

Masters, B. R.

K. H. Kim, C. Buehler, C.-Y. Dong, B. R. Masters, P. T. C. So, “Tissue imaging using two-photon video rate microscopy,” in Optical Diagnostics of Living Cells II, D. L. Farkas, R. C. Leif, B. J. Tromberg, eds., Proc. SPIE3604, 60–66 (1999).
[CrossRef]

Milner, T.

D. Smithies, T. Lindmo, Z. Chen, J. Nelson, T. Milner, “Signal attenuation and localization in optical coherence tomography studied by Monte Carlo simulation,” Phys. Med. Biol. 43, 3025–3044 (1998).
[CrossRef] [PubMed]

Mitra, P.

D. Kleinfeld, P. Mitra, F. Helmchen, W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. USA 95, 15741–15746 (1998).
[CrossRef] [PubMed]

Moes, C.

Nelson, J.

D. Smithies, T. Lindmo, Z. Chen, J. Nelson, T. Milner, “Signal attenuation and localization in optical coherence tomography studied by Monte Carlo simulation,” Phys. Med. Biol. 43, 3025–3044 (1998).
[CrossRef] [PubMed]

Oh, D.

D. Oh, R. Stanley, M. Lin, W. Hoeffler, S. Boxer, M. Berns, E. Bauer, “Two-photon excitation of 4′-Hydroxylmethyl-4,5′,8-Trimethylpsoralen,” Photochem. Photobiol. 65, 91–95 (1997).
[CrossRef] [PubMed]

Olivio, M.

T. Farrel, B. Wilson, M. Patterson, M. Olivio, “Comparison of the in vivo photodynamic threshold dose for photofrin, mono- and tetrasulfonated aluminum phthalocyanine using a rat liver model,” Photochem. Photobiol. 68, 394–399 (1998).
[CrossRef]

Patterson, M.

T. Farrel, B. Wilson, M. Patterson, M. Olivio, “Comparison of the in vivo photodynamic threshold dose for photofrin, mono- and tetrasulfonated aluminum phthalocyanine using a rat liver model,” Photochem. Photobiol. 68, 394–399 (1998).
[CrossRef]

Pham, T.

R. Hornung, T. Pham, K. Keefe, J. Fishkin, M. Berns, Y. Tadir, B. Tromberg, “Quantitative near infrared spectroscopy of cervical dysplasia in vivo,” Hum. Reprod. 14, 2908–2916 (1999).
[CrossRef] [PubMed]

Prahl, S.

H. van Staveren, C. Moes, J. van Marle, S. Prahl, M. van Gemert, “Light scattering in Intralipid-10% in the wavelength range of 400–1100 nm,” Appl. Opt. 30, 4507–4514 (1991).
[CrossRef] [PubMed]

S. Jacques, C. Alter, S. Prahl, “Angular dependence of HeNe laser light scattering by human dermis,” Lasers Life Sci. 1, 309–333 (1987).

Richards-Kortum, R.

Schilders, S.

Schmitt, J.

Sheppard, C.

C. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).

Smithies, D.

D. Smithies, T. Lindmo, Z. Chen, J. Nelson, T. Milner, “Signal attenuation and localization in optical coherence tomography studied by Monte Carlo simulation,” Phys. Med. Biol. 43, 3025–3044 (1998).
[CrossRef] [PubMed]

Smithpeter, C.

Smithpeter, C. L.

So, P. T. C.

K. H. Kim, C. Buehler, C.-Y. Dong, B. R. Masters, P. T. C. So, “Tissue imaging using two-photon video rate microscopy,” in Optical Diagnostics of Living Cells II, D. L. Farkas, R. C. Leif, B. J. Tromberg, eds., Proc. SPIE3604, 60–66 (1999).
[CrossRef]

Stanley, R.

D. Oh, R. Stanley, M. Lin, W. Hoeffler, S. Boxer, M. Berns, E. Bauer, “Two-photon excitation of 4′-Hydroxylmethyl-4,5′,8-Trimethylpsoralen,” Photochem. Photobiol. 65, 91–95 (1997).
[CrossRef] [PubMed]

Strickler, J.

W. Denk, J. Strickler, W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 78–76 (1990).
[CrossRef]

Tadir, Y.

R. Hornung, T. Pham, K. Keefe, J. Fishkin, M. Berns, Y. Tadir, B. Tromberg, “Quantitative near infrared spectroscopy of cervical dysplasia in vivo,” Hum. Reprod. 14, 2908–2916 (1999).
[CrossRef] [PubMed]

Tromberg, B.

R. Hornung, T. Pham, K. Keefe, J. Fishkin, M. Berns, Y. Tadir, B. Tromberg, “Quantitative near infrared spectroscopy of cervical dysplasia in vivo,” Hum. Reprod. 14, 2908–2916 (1999).
[CrossRef] [PubMed]

J. Fishkin, O. Coquoz, E. Anderson, M. Brenner, B. Tromberg, “Frequency-domain photon migration measurements of normal and malignant tissue optical properties in a human subject,” Appl. Opt. 36, 10–20 (1997).
[CrossRef] [PubMed]

van Gemert, M.

van Marle, J.

van Staveren, H.

Webb, W.

Welch, A. J.

White, J.

V. Centonze, J. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75, 2015–2024 (1998).
[CrossRef] [PubMed]

Wilson, B.

T. Farrel, B. Wilson, M. Patterson, M. Olivio, “Comparison of the in vivo photodynamic threshold dose for photofrin, mono- and tetrasulfonated aluminum phthalocyanine using a rat liver model,” Photochem. Photobiol. 68, 394–399 (1998).
[CrossRef]

Xu, C.

Yadlowski, M.

Appl. Opt. (4)

Biophys. J. (1)

V. Centonze, J. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75, 2015–2024 (1998).
[CrossRef] [PubMed]

Hum. Reprod. (1)

R. Hornung, T. Pham, K. Keefe, J. Fishkin, M. Berns, Y. Tadir, B. Tromberg, “Quantitative near infrared spectroscopy of cervical dysplasia in vivo,” Hum. Reprod. 14, 2908–2916 (1999).
[CrossRef] [PubMed]

IEEE J. Sel. Top. Quantum Electron. (1)

A. Dunn, R. Richards-Kortum, “Three-dimensional computation of light scattering from cells,” IEEE J. Sel. Top. Quantum Electron. 2, 898–905 (1997).
[CrossRef]

J. Opt. Soc. Am. A (2)

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

Lasers Life Sci. (1)

S. Jacques, C. Alter, S. Prahl, “Angular dependence of HeNe laser light scattering by human dermis,” Lasers Life Sci. 1, 309–333 (1987).

Opt. Lett. (1)

Optik (1)

C. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).

Photochem. Photobiol. (2)

T. Farrel, B. Wilson, M. Patterson, M. Olivio, “Comparison of the in vivo photodynamic threshold dose for photofrin, mono- and tetrasulfonated aluminum phthalocyanine using a rat liver model,” Photochem. Photobiol. 68, 394–399 (1998).
[CrossRef]

D. Oh, R. Stanley, M. Lin, W. Hoeffler, S. Boxer, M. Berns, E. Bauer, “Two-photon excitation of 4′-Hydroxylmethyl-4,5′,8-Trimethylpsoralen,” Photochem. Photobiol. 65, 91–95 (1997).
[CrossRef] [PubMed]

Phys. Med. Biol. (1)

D. Smithies, T. Lindmo, Z. Chen, J. Nelson, T. Milner, “Signal attenuation and localization in optical coherence tomography studied by Monte Carlo simulation,” Phys. Med. Biol. 43, 3025–3044 (1998).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. USA (1)

D. Kleinfeld, P. Mitra, F. Helmchen, W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. USA 95, 15741–15746 (1998).
[CrossRef] [PubMed]

Science (1)

W. Denk, J. Strickler, W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 78–76 (1990).
[CrossRef]

Other (2)

K. H. Kim, C. Buehler, C.-Y. Dong, B. R. Masters, P. T. C. So, “Tissue imaging using two-photon video rate microscopy,” in Optical Diagnostics of Living Cells II, D. L. Farkas, R. C. Leif, B. J. Tromberg, eds., Proc. SPIE3604, 60–66 (1999).
[CrossRef]

H. Kume, ed., Hamamatsu Photonics, in Photomultiplier Tube: Principle to Application, (Hamamatsu Photonics, Bridgewater, N.J., 1994).

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Figures (7)

Fig. 1
Fig. 1

Geometry used in the Monte Carlo simulation.

Fig. 2
Fig. 2

Fluorescence generation (top) and collection (bottom) at three different scattering coefficients (5, 10, and 15 mm-1). Solid curves, exponential fits to the data.

Fig. 3
Fig. 3

Effect of anisotropy, g, on the generation of two-photon fluorescence at a focus depth of 200 µm.

Fig. 4
Fig. 4

Total detected fluorescence intensity as a function of objective N.A. for focal depths of 100, 200, and 300 µm.

Fig. 5
Fig. 5

Measurement of the lateral PSF of the system. (a) Image of the tissue phantom at a depth of approximately 100 µm. (b) Image of an individual sphere from the image in (a). (c) Gaussian fit to the intensity of a single sphere.

Fig. 6
Fig. 6

Measured two-photon fluorescence as a function of focal depth for 0.1-µm fluorescent spheres embedded in a 2% intralipid phantom (μ s ex = 6 mm-1, μ s em = 16 mm-1).

Fig. 7
Fig. 7

Measured width of 0.1-µm fluorescent spheres embedded in 2% intralipid as a function of focal depth. The width at each depth is the average width of approximately 25 spheres as measured by Gaussian fits to the individual spheres.

Tables (2)

Tables Icon

Table 1 Optical Properties of the 2% Intralipid Agarose Gel Used in the Measurements and Simulations at the Excitation and Emission Wavelengthsa

Tables Icon

Table 2 Optical Properties of Cervical Epithelium Used in the Estimation of Imaging Deptha

Equations (5)

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

Iexr, t=- Gr, tft-tdt,
Fexr=12 ϕσCr- Iex2r, tdt,
Szf=η  FexrFemrdr,
Szf=So exp-bexμtex+bemμtemzf,
SNR=NstNs+2Nd+Nb1/2,

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