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Applied Optics

Applied Optics

APPLICATIONS-CENTERED RESEARCH IN 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)
http://dx.doi.org/10.1364/AO.41.005376


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Abstract

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

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

Citation
Emmanuel Beaurepaire and Jerome Mertz, "Epifluorescence collection in two-photon microscopy," Appl. Opt. 41, 5376-5382 (2002)
http://www.opticsinfobase.org/ao/abstract.cfm?URI=ao-41-25-5376

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