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

Xu, Fang and Wei, Lu and Chen, Zhixing and Min, Wei (2013) Frustrated FRET for high-contrast high-resolution two-photon imaging. Optics Express, 21 (12). pp. 14097-14108. ISSN 1094-4087. doi:10.1364/oe.21.014097.

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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.

Item Type:Article
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URLURL TypeDescription
Wei, Lu0000-0001-9170-2283
Min, Wei0000-0003-2570-3557
Additional Information:© 2013 Optical Society of America. Received 20 Feb 2013; revised 1 Apr 2013; accepted 2 Apr 2013; published 5 Jun 2013. We thank L. Zhang, X. Zhu, L. Brus, R. Yuste, D. Peterka, V. Cornish and M. Jimenez for helpful discussions. W.M. acknowledges support from Kavli Institute for Brain Science and RISE program of Columbia University.
Funding AgencyGrant Number
Kavli Institute for Brain ScienceUNSPECIFIED
Columbia UniversityUNSPECIFIED
Issue or Number:12
Classification Code:OCIS codes: (170.2520) Fluorescence microscopy; (170.4090) Modulation techniques; (170.5810) Scanning microscopy; (180.4315) Nonlinear microscopy; (190.4180) Multiphoton processes; (290.0290) Scattering
Record Number:CaltechAUTHORS:20181119-102039911
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Official 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)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:91014
Deposited By: George Porter
Deposited On:19 Nov 2018 19:11
Last Modified:16 Nov 2021 03:37

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