OSA's Digital Library

Applied Optics

Applied Optics


  • Vol. 39, Iss. 7 — Mar. 1, 2000
  • pp: 1194–1201

Influence of optical properties on two-photon fluorescence imaging in turbid samples

Andrew K. Dunn, Vincent P. Wallace, Mariah Coleno, Michael W. Berns, and Bruce J. Tromberg  »View Author Affiliations

Applied Optics, Vol. 39, Issue 7, pp. 1194-1201 (2000)

View Full Text Article

Acrobat PDF (1618 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



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, <i>g</i>, except for <i>g</i> > 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

OCIS Codes
(170.0180) Medical optics and biotechnology : Microscopy
(170.3660) Medical optics and biotechnology : Light propagation in tissues
(180.0180) Microscopy : Microscopy

Andrew K. Dunn, Vincent P. Wallace, Mariah Coleno, Michael W. Berns, and Bruce J. Tromberg, "Influence of optical properties on two-photon fluorescence imaging in turbid samples," Appl. Opt. 39, 1194-1201 (2000)

Sort:  Author  |  Year  |  Journal  |  Reset


  1. W. Denk, J. Strickler, and W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 78–76 (1990).
  2. K. H. Kim, C. Buehler, C.-Y. Dong, B. R. Masters, and 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, and B. J. Tromberg, eds., Proc. SPIE 3604, 60–66 (1999).
  3. D. Kleinfeld, P. Mitra, F. Helmchen, and 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).
  4. J. Schmitt, A. Knuttel, and M. Yadlowski, “Confocal microscopy in turbid media,” J. Opt. Soc. Am. A 11, 2226–2235 (1994).
  5. X. Gan, S. Schilders, and M. Gu, “Image formation in turbid media under a microscope,” J. Opt. Soc. Am. A 15, 2052–2058 (1998).
  6. A. Dunn, C. Smithpeter, R. Richards-Kortum, and A. J. Welch, “Sources of contrast in confocal reflectance imaging,” Appl. Opt. 35, 3441–3446 (1996).
  7. D. Smithies, T. Lindmo, Z. Chen, J. Nelson, and T. Milner, “Signal attenuation and localization in optical coherence tomography studied by Monte Carlo simulation,” Phys. Med. Biol. 43, 3025–3044 (1998).
  8. X. Gan and M. Gu, “Effective point-spread function for fast image modeling and processing in microscopic imaging through turbid media,” Opt. Lett. 24, 741–743 (1999).
  9. C. Xu and 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).
  10. J. Fishkin, O. Coquoz, E. Anderson, M. Brenner, and B. Tromberg, “Frequency-domain photon migration measurements of normal and malignant tissue optical properties in a human subject,” Appl. Opt. 36, 10–20 (1997).
  11. C. Sheppard and M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).
  12. S. Jacques, C. Alter, and S. Prahl, “Angular dependence of HeNe laser light scattering by human dermis,” Lasers Life Sci. 1, 309–333 (1987).
  13. A. Dunn and R. Richards-Kortum, “Three-dimensional computation of light scattering from cells,” IEEE J. Sel. Top. Quantum Electron. 2, 898–905 (1997).
  14. H. van Staveren, C. Moes, J. van Marle, S. Prahl, and M. van Gemert, “Light scattering in Intralipid-10% in the wavelength range of 400–1100 nm,” Appl. Opt. 30, 4507–4514 (1991).
  15. C. L. Smithpeter, A. K. Dunn, A. J. Welch, and R. Richards-Kortum, “Penetration depth limits of in vivo confocal reflectance imaging,” Appl. Opt. 37, 2749–2754 (1998).
  16. V. Centonze and J. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75, 2015–2024 (1998).
  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, and B. Tromberg, “Quantitative near infrared spectroscopy of cervical dysplasia in vivo,” Hum. Reprod. 14, 2908–2916 (1999).
  19. T. Farrel, B. Wilson, M. Patterson, and 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).
  20. D. Oh, R. Stanley, M. Lin, W. Hoeffler, S. Boxer, M. Berns, and E. Bauer, “Two-photon excitation of 4′-Hydroxylmethyl-4,5′, 8-Trimethylpsoralen,” Photochem. Photobiol. 65, 91–95 (1997).

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited