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Applied Optics

Applied Optics


  • Vol. 41, Iss. 25 — Sep. 1, 2002
  • pp: 5376–5382

Epifluorescence collection in two-photon microscopy

Emmanuel Beaurepaire and Jerome Mertz  »View Author Affiliations

Applied Optics, Vol. 41, Issue 25, pp. 5376-5382 (2002)

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We present a simple model to describe epifluorescence collection in two-photon microscopy when one images in a turbid slab with an objective. Bulk and surface scattering determine the spatial and angular distributions of the outgoing fluorescence photons at the slab surface, and geometrical optics determines how efficiently the photons are collected. The collection optics are parameterized by the objective’s numerical aperture and working distance and by an effective collection field of view. We identify the roles of each of these parameters and provide simple rules of thumb for the optimization of the epifluorescence collection efficiency. Analytical results are corroborated by Monte Carlo simulation.

© 2002 Optical Society of America

OCIS Codes
(170.3660) Medical optics and biotechnology : Light propagation in tissues
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(180.2520) Microscopy : Fluorescence microscopy
(180.5810) Microscopy : Scanning microscopy
(180.6900) Microscopy : Three-dimensional microscopy
(190.4180) Nonlinear optics : Multiphoton processes

Original Manuscript: January 9, 2002
Revised Manuscript: March 29, 2002
Published: September 1, 2002

Emmanuel Beaurepaire and Jerome Mertz, "Epifluorescence collection in two-photon microscopy," Appl. Opt. 41, 5376-5382 (2002)

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  1. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990). [CrossRef] [PubMed]
  2. 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]
  3. E. Beaurepaire, M. Oheim, J. Mertz, “Ultra-deep two-photon fluorescence excitation in turbid media,” Opt. Commun. 188, 25–29 (2001). [CrossRef]
  4. C. M. Blanca, C. Saloma, “Monte Carlo analysis of two-photon fluorescence imaging through a scattering medium,” Appl. Opt. 37, 8092–8102 (1998). [CrossRef]
  5. J. Ying, F. Liu, R. R. Alfano, “Effect of scattering on nonlinear optical scanning microscopy imaging of highly scattering media,” Appl. Opt. 39, 509–514 (2000). [CrossRef]
  6. A. K. Dunn, V. P. Wallace, M. Coleno, M. W. Berns, B. J. Tromberg, “Influence of optical properties on two-photon fluorescence imaging in turbid samples,” Appl. Opt. 39, 1194–1201 (2000). [CrossRef]
  7. M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods 112, 205 (2001). [CrossRef]
  8. V. Tuchin, Tissue Optics (SPIE, Bellingham, Washington, 2000).
  9. R. C. Haskell, L. O. Svaasand, T.-T. Tsay, T.-C. Feng, M. S. McAdams, B. J. Tromberg, “Boundary conditions for the diffusion equation in radiative transfer,” J. Opt. Soc. A 11, 2727–2741 (1994). [CrossRef]
  10. M. S. Patterson, B. Chance, B. C. Wilson, “Time resolved reflectance and transmittance for the noninvasive measurement of tissue optical properties,” Appl. Opt. 28, 2331–2336 (1989). [CrossRef] [PubMed]
  11. K. M. Case, P. F. Zweifel, Linear Transport Theory (Addison-Wesley, Reading, Mass., 1967).
  12. H. S. Carslaw, J. C. Jaeger, Conduction of Heat in Solids, 2nd ed. (Oxford Science, Oxford, 1996).
  13. L. Henyey, J. Greenstein, “Diffuse radiation in the galaxy,” Astrophys. J. 93, 70–83 (1941). [CrossRef]
  14. S. A. Prahl, M. Keijzer, S. L. Jacques, A. J. Welch, “A Monte Carlo model of light propagation in tissue,” in SPIE Proceedings of Dosimetry of Laser Radiation in Medicine and Biology, G. J. Mueller, D. H. Sliney, eds., Vol. IS05 of SPIE Institute Series (SPIE, Bellingham, Wash., 1989), pp. 102–111.

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