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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 3, Iss. 8 — Aug. 1, 2012
  • pp: 1955–1963

Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast

Ya-Ting Kao, Xinxin Zhu, Fang Xu, and Wei Min  »View Author Affiliations

Biomedical Optics Express, Vol. 3, Issue 8, pp. 1955-1963 (2012)

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Probing biological structures and functions deep inside live organisms with light is highly desirable. Among the current optical imaging modalities, multiphoton fluorescence microscopy exhibits the best contrast for imaging scattering samples by employing a spatially confined nonlinear excitation. However, as the incident laser power drops exponentially with imaging depth into the sample due to the scattering loss, the out-of-focus background eventually overwhelms the in-focus signal, which defines a fundamental imaging-depth limit. Herein we significantly improve the image contrast for deep scattering samples by harnessing reversibly switchable fluorescent proteins (RSFPs) which can be cycled between bright and dark states upon light illumination. Two distinct techniques, multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI), are demonstrated on tissue phantoms labeled with Dronpa protein. Such a focal switch approach can generate pseudo background-free images. Conceptually different from wave-based approaches that try to reduce light scattering in turbid samples, our work represents a molecule-based strategy that focused on imaging probes.

© 2012 OSA

OCIS Codes
(180.2520) Microscopy : Fluorescence microscopy
(190.4180) Nonlinear optics : Multiphoton processes
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:

Original Manuscript: May 30, 2012
Revised Manuscript: June 17, 2012
Manuscript Accepted: June 19, 2012
Published: July 27, 2012

Ya-Ting Kao, Xinxin Zhu, Fang Xu, and Wei Min, "Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast," Biomed. Opt. Express 3, 1955-1963 (2012)

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