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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 20, Iss. 17 — Aug. 13, 2012
  • pp: 18525–18536

Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores

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

Optics Express, Vol. 20, Issue 17, pp. 18525-18536 (2012)

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It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP.

© 2012 OSA

OCIS Codes
(160.4330) Materials : Nonlinear optical materials
(170.5810) Medical optics and biotechnology : Scanning microscopy
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence
(180.2520) Microscopy : Fluorescence microscopy
(190.4180) Nonlinear optics : Multiphoton processes
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:

Original Manuscript: April 12, 2012
Revised Manuscript: June 18, 2012
Manuscript Accepted: June 19, 2012
Published: July 30, 2012

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

Zhixing Chen, Lu Wei, Xinxin Zhu, and Wei Min, "Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores," Opt. Express 20, 18525-18536 (2012)

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