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Applied Optics

Applied Optics


  • Vol. 39, Iss. 28 — Oct. 1, 2000
  • pp: 5244–5255

Excitation with a focused, pulsed optical beam in scattering media: diffraction effects

Vincent Ricardo Daria, Caesar Saloma, and Satoshi Kawata  »View Author Affiliations

Applied Optics, Vol. 39, Issue 28, pp. 5244-5255 (2000)

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To gain a better understanding of the spatiotemporal problems that are encountered in two-photon excitation fluorescence imaging through highly scattering media, we investigate how diffraction affects the three-dimensional intensity distribution of a focused, pulsed optical beam propagating inside a scattering medium. In practice, the full potential of the two-photon excitation fluorescence imaging is unrealized at long scattering depths, owing to the unwanted temporal and spatial broadening of the femtosecond excitation light pulse that reduces the energy density at the geometric focus while it increases the excitation energy density in the out-of-focus regions. To analyze the excitation intensity distribution, we modify the Monte Carlo–based photon-transport model to a semi-quantum-mechanical representation that combines the wave properties of light with the particle behavior of the propagating photons. In our model the propagating photon is represented by a plane wave with its propagation direction in the scattering medium determined by the Monte Carlo technique. The intensity distribution in the focal region is given by the square of the linear superposition of the various plane waves that arrive at different incident angles and optical path lengths. In the absence of scattering, the propagation model yields the intensity distribution that is predicted by the Huygens–Fresnel principle. We quantify the decrease of the energy density delivered at the geometric focus as a function of the optical depth to the mean-free-path ratio that yields the average number of scattering events that a photon encounters as it propagates toward the focus. Both isotropic and anisotropic scattering media are considered. Three values for the numerical aperture (NA) of the focusing lens are considered: NA = 0.25, 0.5, 0.75.

© 2000 Optical Society of America

OCIS Codes
(110.4850) Imaging systems : Optical transfer functions
(180.2520) Microscopy : Fluorescence microscopy
(290.7050) Scattering : Turbid media

Original Manuscript: February 22, 2000
Revised Manuscript: June 27, 2000
Published: October 1, 2000

Vincent Ricardo Daria, Caesar Saloma, and Satoshi Kawata, "Excitation with a focused, pulsed optical beam in scattering media: diffraction effects," Appl. Opt. 39, 5244-5255 (2000)

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  1. C. Saloma, C. Palmes-Saloma, H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in turbid medium,” Phys. Med. Biol. 43, 1741–1759 (1998). [CrossRef] [PubMed]
  2. C. Palmes-Saloma, C. Saloma, “Long-depth imaging of specific gene expressions in whole-mount mouse embryos with single-photon excitation confocal fluorescence microscopy and FISH,” J. Structural Biol. 131, 56–66 (2000). [CrossRef]
  3. C. Blanca, C. Saloma, “Monte Carlo analysis of two-photon fluorescence imaging through a scattering medium,” Appl. Opt. 37, 8092–8102 (1998). [CrossRef]
  4. V. Daria, C. Palmes-Saloma, K. Fujita, C. Saloma, O. Nakamura, H. Kondoh, S. Kawata, “Long depth imaging of turbid biological samples by two-photon microscopy,” in Proceedings of the Nineteenth Meeting of Japan Society for Laser Microscopy (Japan Society for Laser Microscopy, Nagoya, Japan) pp. 28–32.
  5. 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]
  6. V. Daria, O. Nakamura, C. Palmes-Saloma, S. Kawata, “Enhanced depth penetration in imaging of turbid biological samples by two-photon fluorescence microscopy,” Jpn. J. Appl. Phys. 37, 959–961 (1998). [CrossRef]
  7. A. Egner, S. Hell, “Equivalence of the Huygens-Fresnel and Debye approach for the calculation of high aperture point-spread functions in the presence of refractive index mismatch,” J. Microsc. 193, 244–249 (1999). [CrossRef]
  8. O. Nakamura, “Fundamentals of two-photon microscopy,” Microsc. Res. Tech. 47, 165–171 (1999). [CrossRef] [PubMed]
  9. W. Denk, J. Strickler, W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990). [CrossRef] [PubMed]
  10. W. Denk, D. Piston, W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in Handbook of Biological Confocal Microscopy2nd ed., J. Pawley, ed. (Plenum, New York, 1995). [CrossRef]
  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. C. Blanca, C. Saloma, “Efficient analysis of temporal broadening of a pulsed focused Gaussian beam in scattering media,” Appl. Opt. 38, 5433–5437 (1999). [CrossRef]
  13. J. Schmitt, A. Knüttel, M. Yadlowski, “Confocal microscopy in turbid media,” J. Opt. Soc. A 11, 2226–2235 (1994). [CrossRef]
  14. X. Gan, M. Gu, “Spatial distribution of single-photon and two-photon fluorescence light in scattering media: Monte Carlo simulation,” Appl. Opt. 39, 1575–1579 (2000). [CrossRef]
  15. H. van de Hulst, Light Scattering by Small Particles (Dover, New York, 1981).
  16. W. Cheong, S. Prahl, A. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990). [CrossRef]
  17. M. van Rossum and Th. M. Nieuwenhuizen, “Multiple scattering of classical waves: microscopy, mesoscopy, and diffusion,” Rev. Mod. Phys. 71, 313–369 (1999). [CrossRef]
  18. G. J. Tearney, B. E. Bouma, S. A. Boppart, B. Golubovic, E. A. Swanson, J. G. Fujimoto, “Rapid acquisition of in vivo biological images by use of optical coherence tomography,” Opt. Lett. 21, 1408–1410 (1996). [CrossRef] [PubMed]
  19. A. Schonle, S. W. Hell, “Heating by absorption in the focus of an objective lens,” Opt. Lett. 23, 325–327 (1998). [CrossRef]
  20. P. Torok, P. Varga, Z. Laczik, G. Booker, “Electromagnetic diffraction of light focused through a planar interface between materials of mismatched refractive indices: an integral representation,” J. Opt. Soc. Am. A 12, 325–332 (1995). [CrossRef]
  21. P. Torok, P. Varga, Z. Laczik, G. Booker, “Electromagnetic diffraction of light focused through a planar interface between materials of mismatched refractive indices: structure of the electromagnetic field,” J. Opt. Soc. Am. A 12, 2136–2144 (1995). [CrossRef]
  22. S. Hell, G. Reiner, C. Cremer, E. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169, 391–406 (1993). [CrossRef]
  23. T. Wilson, R. Juskaitis, “The axial response of confocal microscopes with high numerical aperture objective lenses,” Bioimaging 3, 35–38 (1995). [CrossRef]
  24. P. Hanninen, S. Hell, “Femtosecond pulse broadening in the focal region of a two-photon fluorescence microscope,” Bioimaging 2, 117–121 (1994). [CrossRef]
  25. M. Born, E. Wolf, Principles of Optics, 7th ed. (Pergamon, Oxford, 1999). [CrossRef]
  26. S. Flock, M. Patterson, B. Wilson, D. Wyman, “Monte Carlo modeling of light propagation in highly scattering tissues: I. Model predictions and comparison with diffusion theory,” IEEE Trans. BioMed. Eng. 26, 1162–1168 (1989). [CrossRef]
  27. J. Goodman, Introduction to Fourier Optics (McGraw-Hill, San Francisco, 1968).
  28. B. Richards, E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. R. Soc. London Ser. A 253, 358–379 (1959). [CrossRef]
  29. E. Goldin, Waves and Photons: An Introduction to Quantum Optics (Wiley, New York, 1982).
  30. M. Havukainen, G. Drobny, S. Stenholm, V. Buzek, “Quantum simulations of optical systems,” J. Mod. Opt. 46, 1343–1367 (1999).
  31. W. Press, S. Teukolsky, W. Vetterling, B. Flannery, Numerical Recipes in C: The Art of Scientific Computing (Cambridge University Press, New York, 1993).
  32. J. Lock, E. Hovenac, “Internal caustic structure of illuminated liquid droplets,” J. Opt. Soc. Am. A 8, 1541–1552 (1991). [CrossRef]
  33. C. Saloma, M. O. Cambaliza, “Single-Gaussian-beam interaction with a dielectric microsphere: radiation forces, multiple internal reflections, and caustic structures,” Appl. Opt. 34, 3522–3528 (1995). [CrossRef] [PubMed]

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