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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics


  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 8, Iss. 7 — Aug. 1, 2013

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)

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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.

© 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

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

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)

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  1. R. Yuste, ed., Imaging: A Laboratory Manual (Cold Spring Harbor Laboratory, 2010).
  2. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990). [CrossRef] [PubMed]
  3. F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods2(12), 932–940 (2005). [CrossRef] [PubMed]
  4. B. R. Master and P. T. C. So, Handbook of Biomedical Nonlinear Optical Microscopy (Oxford University, 2008).
  5. P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A23(12), 3139–3149 (2006). [CrossRef] [PubMed]
  6. P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 μm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett.28(12), 1022–1024 (2003). [CrossRef] [PubMed]
  7. J. Ying, F. Liu, and R. R. Alfano, “Spatial distribution of two-photon-excited fluorescence in scattering media,” Appl. Opt.38(1), 224–229 (1999). [CrossRef] [PubMed]
  8. N. J. Durr, C. T. Weisspfennig, B. A. Holfeld, and A. Ben-Yakar, “Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues,” J. Biomed. Opt.16(2), 026008 (2011). [CrossRef] [PubMed]
  9. D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt.16(10), 106014 (2011). [CrossRef] [PubMed]
  10. N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods7(2), 141–147 (2010). [CrossRef] [PubMed]
  11. M. Rueckel, J. A. Mack-Bucher, and W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Natl. Acad. Sci. U.S.A.103(46), 17137–17142 (2006). [CrossRef] [PubMed]
  12. A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J.94(4), 1449–1458 (2008). [CrossRef] [PubMed]
  13. M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett.22(24), 1905–1907 (1997). [CrossRef] [PubMed]
  14. Z. Yaqoob, D. Psaltis, M. S. Feld, and C. Yang, “Optical phase conjugation for turbidity suppression in biological samples,” Nat. Photonics2(2), 110–115 (2008). [CrossRef] [PubMed]
  15. G. Zhu, J. van Howe, M. Durst, W. Zipfel, and C. Xu, Simultaneous Spatial and Temporal Focusing of Femtosecond Pulses (Conference on Lasers and Electro-Optics (CLEO), 2005). [CrossRef]
  16. N. Chen, C. H. Wong, and C. J. Sheppard, “Focal modulation microscopy,” Opt. Express16(23), 18764–18769 (2008). [CrossRef] [PubMed]
  17. J. B. Ding, K. T. Takasaki, and B. L. Sabatini, “Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy,” Neuron63(4), 429–437 (2009). [CrossRef] [PubMed]
  18. P. Bianchini, B. Harke, S. Galiani, G. Vicidomini, and A. Diaspro, “Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging,” Proc. Natl. Acad. Sci. U.S.A.109(17), 6390–6393 (2012). [CrossRef] [PubMed]
  19. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006). [CrossRef] [PubMed]
  20. M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods3(10), 793–796 (2006). [CrossRef] [PubMed]
  21. M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A.102(37), 13081–13086 (2005). [CrossRef] [PubMed]
  22. T. Dertinger, R. Colyer, G. Iyer, S. Weiss, and J. Enderlein, “Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI),” Proc. Natl. Acad. Sci. U.S.A.106(52), 22287–22292 (2009). [CrossRef] [PubMed]
  23. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett.19(11), 780–782 (1994). [CrossRef] [PubMed]
  24. S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a living mouse brain,” Science335(6068), 551 (2012). [CrossRef] [PubMed]
  25. M. Beutler, K. Makrogianneli, R. J. Vermeij, M. Keppler, T. Ng, T. M. Jovin, and R. Heintzmann, “satFRET: estimation of Forster resonance energy transfer by acceptor saturation,” Eur. Biophys. J.38(1), 69–82 (2008). [CrossRef] [PubMed]
  26. P. E. Hänninen, L. Lehtelä, and S. W. Hell, “Two- and multiphoton excitation of conjugate-dyes using a continuous wave laser,” Opt. Commun.130(1-3), 29–33 (1996). [CrossRef]
  27. A. Schönle, P. E. Hanninen, and S. W. Hell, “Nonlinear fluorescence through intermolecular energy transfer and resolution increase in fluorescence microscopy,” Ann. Phys.8(2), 115–133 (1999). [CrossRef]
  28. C. I. Richards, J. C. Hsiang, A. M. Khalil, N. P. Hull, and R. M. Dickson, “FRET-enabled optical modulation for high sensitivity fluorescence imaging,” J. Am. Chem. Soc.132(18), 6318–6323 (2010). [CrossRef] [PubMed]
  29. K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett.99(22), 228105 (2007). [CrossRef] [PubMed]
  30. J. Lukomska, I. Gryczynski, J. Malicka, S. Makowiec, J. R. Lakowicz, and Z. Gryczynski, “Two-photon induced fluorescence of Cy5-DNA in buffer solution and on silver island films,” Biochem. Biophys. Res. Commun.328(1), 78–84 (2005). [CrossRef] [PubMed]
  31. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol.21(11), 1369–1377 (2003). [CrossRef] [PubMed]
  32. Y.-T. Kao, X. Zhu, F. Xu, and W. Min, “Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast,” Biomed. Opt. Express3(8), 1955–1963 (2012). [CrossRef] [PubMed]
  33. Z. Chen, L. Wei, X. Zhu, and W. Min, “Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores,” Opt. Express20(17), 18525–18536 (2012). [CrossRef] [PubMed]
  34. X. Zhu, Y.-T. Kao, and W. Min, “Molecular-switch-mediated multiphoton fluorescence microscopy with high-order nonlinearity,” J. Phys. Chem. Lett.3(15), 2082–2086 (2012). [CrossRef]

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