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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 20, Iss. 18 — Aug. 27, 2012
  • pp: 20605–20622

Enhancement of imaging depth of two-photon microscopy using pinholes: Analytical simulation and experiments

Woosub Song, Jihoon Lee, and Hyuk-Sang Kwon  »View Author Affiliations

Optics Express, Vol. 20, Issue 18, pp. 20605-20622 (2012)

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Achieving a greater imaging depth with two-photon fluorescence microscopy (TPFM) is mainly limited by out-of-focus fluorescence generated from both ballistic and scattered light excitation. We report on an improved signal-to-noise ratio (SNR) in a highly scattering medium as demonstrated by analytical simulation and experiments for TPFM. Our technique is based on out-of-focus rejection using a confocal pinhole. We improved the SNR by introducing the pinhole in the collection beam path. Using the radiative transfer theory and the ray-optics approach, we analyzed the effects of different sizes of pinholes on the generation of the fluorescent signal in the TPFM system. The analytical simulation was evaluated by comparing its results with the experimental results in a scattering medium. In a combined confocal pinhole and two-photon microscopy system, the imaging depth limit of approximately 5 scattering mean free paths (MFP) was found to have improved to 6.2 MFP.

© 2012 OSA

OCIS Codes
(110.2990) Imaging systems : Image formation theory
(170.3660) Medical optics and biotechnology : Light propagation in tissues
(190.4180) Nonlinear optics : Multiphoton processes
(110.0113) Imaging systems : Imaging through turbid media
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:

Original Manuscript: June 18, 2012
Revised Manuscript: August 13, 2012
Manuscript Accepted: August 15, 2012
Published: August 23, 2012

Virtual Issues
Vol. 7, Iss. 10 Virtual Journal for Biomedical Optics

Woosub Song, Jihoon Lee, and Hyuk-Sang Kwon, "Enhancement of imaging depth of two-photon microscopy using pinholes: Analytical simulation and experiments," Opt. Express 20, 20605-20622 (2012)

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