OSA's Digital Library

Applied Optics

Applied Optics


  • Vol. 37, Iss. 34 — Dec. 1, 1998
  • pp: 8092–8102

Monte Carlo analysis of two-photon fluorescence imaging through a scattering medium

Carlo Mar Blanca and Caesar Saloma  »View Author Affiliations

Applied Optics, Vol. 37, Issue 34, pp. 8092-8102 (1998)

View Full Text Article

Enhanced HTML    Acrobat PDF (226 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



The behavior of two-photon fluorescence imaging through a scattering medium is analyzed by use of the Monte Carlo technique. The axial and transverse distributions of the excitation photons in the focused Gaussian beam are derived for both isotropic and anisotropic scatterers at different numerical apertures and at various ratios of the scattering depth with the mean free path. The two-photon fluorescence profiles of the sample are determined from the square of the normalized excitation intensity distributions. For the same lens aperture and scattering medium, two-photon fluorescence imaging offers a sharper and less aberrated axial response than that of single-photon confocal fluorescence imaging. The contrast in the corresponding transverse fluorescence profile is also significantly higher. Also presented are results comparing the effects of isotropic and anisotropic scattering media in confocal reflection imaging. The convergence properties of the Monte Carlo simulation are also discussed.

© 1998 Optical Society of America

OCIS Codes
(170.1790) Medical optics and biotechnology : Confocal microscopy
(170.2520) Medical optics and biotechnology : Fluorescence microscopy

Original Manuscript: May 26, 1998
Revised Manuscript: August 3, 1998
Published: December 1, 1998

Carlo Mar Blanca and Caesar Saloma, "Monte Carlo analysis of two-photon fluorescence imaging through a scattering medium," Appl. Opt. 37, 8092-8102 (1998)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. B. Rosen, R. Beddington, “Whole-mount hybridization in the mouse embryo: gene expression in three dimensions,” Trends Genet. 9, 162–163 (1993). [CrossRef] [PubMed]
  2. D. Wilkinson, In Situ Hybridization: A Practical Approach (International Reproduction Ltd., New York, 1993), pp. 75–83.
  3. R. Haugland, Handbook in Fluorescent Probes and Research Chemicals, 6th ed. (Molecular Probes, Eugene, Ore., 1996).
  4. C. Saloma, C. Palmes-Saloma, H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in a turbid medium,” Phys. Med. Biol. 43, 1741–1759 (1998). [CrossRef] [PubMed]
  5. W. Denk, J. Strickler, W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990). [CrossRef] [PubMed]
  6. W. Denk, K. Delaney, A. Gelperin, D. Kleinfeld, B. Strowbridge, D. Tank, R. Yuste, “Anatomical and functional imaging of neurons using two-photon laser scanning microscopy,” J. Neurosci. Methods 54, 151–162 (1994). [CrossRef] [PubMed]
  7. D. Piston, M. S. Kirby, H. Cheng, W. Lederer, W. Webb, “Two-photon-excitation fluorescence imaging of three-dimensional calcium-ion activity,” Appl. Opt. 33, 662–669 (1994). [CrossRef] [PubMed]
  8. R. Yuste, W. Denk, “Dendritic spines as basic functional units of neuronal integration,” Nature 375, 682–684 (1995). [CrossRef] [PubMed]
  9. J. Manni, “Two-photon excitation expands the capabilities of laser-scanning microscopy,” Biophotonics 1996, 44–49.
  10. K. Svoboda, W. Denk, S. Tsuda, “Two-photon excitation scanning microscopy of living neurons with a saturable Bragg reflector mode-locked diode pumped Cr:LiSrAlFl laser,” Opt. Lett. 21, 1411–1413 (1996). [CrossRef] [PubMed]
  11. 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]
  12. W. Fisher, E. Wachter, M. Armas, C. Seaton, “Ti:sapphire laser as an excitation source in two-photon spectroscopy,” Appl. Spectrosc. 51, 218–226 (1997). [CrossRef]
  13. S. Potter, “Vital imaging: two photons are better than one,” Current Biol. 6, 1595–1598 (1996). [CrossRef]
  14. W. Denk, K. Svoboda, “Photon upmanship: why multiphoton is more than a gimmick,” Neuron 18, 351–357 (1997). [CrossRef] [PubMed]
  15. C. Sheppard, K. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).
  16. O. Nakamura, “Three-dimensional imaging characteristics of laser scan fluorescence microscopy: two-photon excitation versus single-photon excitation,” Optik 93, 39–42 (1993).
  17. J. Schmitt, A. Knuttel, M. Yadlowsky, “Confocal microscopy in turbid media,” J. Opt. Soc. Am. A 11, 2226–2235 (1994). [CrossRef]
  18. J. Schmitt, K. Ben-Letaief, “Efficient Monte Carlo simulation of confocal microscopy in biological tissue,” J. Opt. Soc. Am. A 13, 952–961 (1996). [CrossRef]
  19. A. Dunn, C. Smithpeter, A. Welch, R. Richards-Kortum, “Sources of contrast in confocal reflectance imaging,” Appl. Opt. 35, 3441–3446 (1996). [CrossRef] [PubMed]
  20. H. C. van de Hulst, Multiple Scattering Light Tables, Formulas and Applications (Academic, New York, 1980).
  21. M. Born, E. Wolf, Principles of Optics (Pergamon, New York, 1991).
  22. M. Cambaliza, C. Saloma, “Single Gaussian beam interaction with a dielectric microsphere: radiation forces, multiple internal reflections, and caustic structures,” Appl. Opt. 34, 3522–3528 (1995). [CrossRef]
  23. S. T. Flock, M. S. Patterson, B. C. Wilson, D. R. Wyman, “Monte Carlo modeling of light propagation in highly scattering tissues—I,” IEEE Trans. Biomed. Eng. 36, 1162–1167 (1989). [CrossRef] [PubMed]
  24. R. Newton, Scattering Theory of Waves and Particles (McGraw-Hill, New York, 1966).
  25. A. Seigman, Lasers (University Science, Mill Valley, Calif., 1986), Chap. 17.
  26. J. Proakis, D. Manolakis, Introduction to Digital Signal Processing (Macmillan, New York, 1989), pp. 129–133.
  27. S. Flock, B. Wilson, M. Patterson, “Total attenuation coefficients and scattering phase functions of tissues and phantom materials at 633 nm,” Med. Phys. 14, 835–841 (1987). [CrossRef] [PubMed]
  28. S. Jacques, C. Alter, S. Prahl, “Angular dependence of HeNe laser light scattering by human dermis,” Laser Life Sci. 1, 309–333 (1987).
  29. W. Cheong, S. Prahl, A. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990). [CrossRef]
  30. M. Gu, “Resolution in three-photon fluorescence scanning microscopy,” Opt. Lett. 21, 988–990 (1996). [CrossRef] [PubMed]
  31. M. Schrader, K. Bahlmann, S. Hell, “Three-photon excitation microscopy: theory, experiment and applications,” Optik 104, 116–121 (1997).
  32. H. Haugen, A. Orthonos, “Fluorescence studies of multiphoton ionization processes: four- and five-photon ionization of Sr at wavelengths of 558–590 nm,” Phys. Rev. A 39, 3392–3399 (1989). [CrossRef] [PubMed]
  33. M. Gu, T. Tannous, C. Sheppard, “Effect of an annular pupil on confocal imaging through highly scattering media,” Opt. Lett. 21, 312–315 (1996). [CrossRef] [PubMed]

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