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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 3, Iss. 6 — Jun. 1, 2012
  • pp: 1465–1475

Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging

Lu Wei, Zhixing Chen, and Wei Min  »View Author Affiliations


Biomedical Optics Express, Vol. 3, Issue 6, pp. 1465-1475 (2012)
http://dx.doi.org/10.1364/BOE.3.001465


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Abstract

Two-photon fluorescence microscopy has become an indispensable tool for imaging scattering biological samples by detecting scattered fluorescence photons generated from a spatially confined excitation volume. However, this optical sectioning capability breaks down eventually when imaging much deeper, as the out-of-focus fluorescence gradually overwhelms the in-focal signal in the scattering samples. The resulting loss of image contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation efficiency. Herein we propose to extend this depth limit by performing stimulated emission reduced fluorescence (SERF) microscopy in which the two-photon excited fluorescence at the focus is preferentially switched on and off by a modulated and focused laser beam that is capable of inducing stimulated emission of the fluorophores from the excited states. The resulting image, constructed from the reduced fluorescence signal, is found to exhibit a significantly improved signal-to-background contrast owing to its overall higher-order nonlinear dependence on the incident laser intensity. We demonstrate this new concept by both analytical theory and numerical simulations. For brain tissues, SERF is expected to extend the imaging depth limit of two-photon fluorescence microscopy by a factor of more than 1.8.

© 2012 OSA

OCIS Codes
(170.4090) Medical optics and biotechnology : Modulation techniques
(180.2520) Microscopy : Fluorescence microscopy
(190.4180) Nonlinear optics : Multiphoton processes
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:
Microscopy

History
Original Manuscript: April 27, 2012
Revised Manuscript: May 19, 2012
Manuscript Accepted: May 19, 2012
Published: May 22, 2012

Citation
Lu Wei, Zhixing Chen, and Wei Min, "Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging," Biomed. Opt. Express 3, 1465-1475 (2012)
http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-3-6-1465


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