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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 21, Iss. 12 — Jun. 17, 2013
  • pp: 14097–14108

Frustrated FRET for high-contrast high-resolution two-photon imaging

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


Optics Express, Vol. 21, Issue 12, pp. 14097-14108 (2013)
http://dx.doi.org/10.1364/OE.21.014097


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Abstract

Two-photon fluorescence microscopy has become increasingly popular in biomedical research as it allows high-resolution imaging of thick biological specimen with superior contrast and penetration than confocal microscopy. However, two-photon microscopy still faces two fundamental limitations: 1) image-contrast deterioration with imaging depth due to out-of-focus background and 2) diffraction-limited spatial resolution. Herein we propose to create and detect high-order (more than quadratic) nonlinear signals by harnessing the frustrated fluorescence resonance energy transfer (FRET) effect within a specially designed donor-acceptor probe pair. Two distinct techniques are described. In the first method, donor fluorescence generated by a two-photon laser at the focus is preferentially switched on and off by a modulated and focused one-photon laser beam that is able to block FRET via direct acceptor excitation. The resulting image, constructed from the enhanced donor fluorescence signal, turns out to be an overall three-photon process. In the second method, a two-photon laser at a proper wavelength is capable of simultaneously exciting both the donor and the acceptor. By sinusoidally modulating the two-photon excitation laser at a fundamental frequency ω, an overall four-photon signal can be isolated by demodulating the donor fluorescence at the third harmonic frequency 3ω. We show that both the image contrast and the spatial resolution of the standard two-photon fluorescence microscopy can be substantially improved by virtue of the high-order nonlinearity. This frustrated FRET approach represents a strategy that is based on extracting the inherent nonlinear photophysical response of the specially designed imaging probes.

© 2013 OSA

OCIS Codes
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(170.4090) Medical optics and biotechnology : Modulation techniques
(170.5810) Medical optics and biotechnology : Scanning microscopy
(190.4180) Nonlinear optics : Multiphoton processes
(290.0290) Scattering : Scattering
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: February 20, 2013
Revised Manuscript: April 1, 2013
Manuscript Accepted: April 2, 2013
Published: June 5, 2013

Virtual Issues
Vol. 8, Iss. 7 Virtual Journal for Biomedical Optics

Citation
Fang Xu, Lu Wei, Zhixing Chen, and Wei Min, "Frustrated FRET for high-contrast high-resolution two-photon imaging," Opt. Express 21, 14097-14108 (2013)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-21-12-14097


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