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Fluorescence imaging beyond the ballistic regime by ultrasound-pulse-guided digital phase conjugation

Abstract

Fluorescence imaging has revolutionized biomedical research over the past three decades. Its high molecular specificity and unrivalled single-molecule-level sensitivity have enabled breakthroughs in a number of research fields. For in vivo applications its major limitation is its superficial imaging depth, a result of random scattering in biological tissues causing exponential attenuation of the ballistic component of a light wave. Here, we present fluorescence imaging beyond the ballistic regime by combining single-cycle pulsed ultrasound modulation and digital optical phase conjugation. We demonstrate a near-isotropic three-dimensional localized sound–light interaction zone. With the exceptionally high optical gain provided by the digital optical phase conjugation system, we can deliver sufficient optical power to a focus inside highly scattering media for not only fluorescence imaging but also a variety of linear and nonlinear spectroscopy measurements. This technology paves the way for many important applications in both fundamental biology research and clinical studies.

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Figure 1: Experimental scheme and set-up.
Figure 2: PSF measurement.
Figure 3: Fluorescence imaging.

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References

  1. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    Article  ADS  Google Scholar 

  2. Planchon, T. A. et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nature Methods 8, 417–U468 (2011).

    Article  Google Scholar 

  3. Wilt, B. A. et al. Advances in light microscopy for neuroscience. Annu. Rev. Neurosci. 32, 435–506 (2009).

    Article  Google Scholar 

  4. Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E. H. K. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

    Article  ADS  Google Scholar 

  5. Theer, P. & Denk, W. On the fundamental imaging-depth limit in two-photon microscopy. J. Opt. Soc. Am. A 23, 3139–3149 (2006).

    Article  ADS  Google Scholar 

  6. Theer, P., Hasan, M. T. & Denk, W. Two-photon imaging to a depth of 1000 µm in living brains by use of a Ti:Al2O3 regenerative amplifier. Opt. Lett. 28, 1022–1024 (2003).

    Article  ADS  Google Scholar 

  7. Tang, J., Germain, R. N. & Cui, M. Superpenetration optical microscopy by iterative multiphoton adaptive compensation technique. Proc. Natl Acad. Sci. 109, 8434–8439 (2012).

    Article  ADS  Google Scholar 

  8. Supatto, W., McMahon, A., Fraser, S. E. & Stathopoulos, A. Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis. Nature Protoc. 4, 1397–1412 (2009).

    Article  Google Scholar 

  9. Mempel, T. R., Henrickson, S. E. & von Andrian, U. H. T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 427, 154–159 (2004).

    Article  ADS  Google Scholar 

  10. Feinberg, J. & Hellwarth, R. W. Phase-conjugating mirror with continous-wave gain. Opt. Lett. 5, 519–521 (1980).

    Article  ADS  Google Scholar 

  11. Yariv, A. & Yeh, P. Phase conjugate optics and real-time holography. IEEE J. Quantum Electron. 14, 650–660 (1978).

    Article  ADS  Google Scholar 

  12. Leith, E. N. & Upatniek, J. Holographic imagery through diffusing media. J. Opt. Soc. Am. 56, 523 (1966).

    Article  Google Scholar 

  13. Vellekoop, I. M. & Mosk, A. P. Universal optimal transmission of light through disordered materials. Phys. Rev. Lett. 101, 120601 (2008).

    Article  ADS  Google Scholar 

  14. Vellekoop, I. M. & Mosk, A. P. Focusing coherent light through opaque strongly scattering media. Opt. Lett. 32, 2309–2311 (2007).

    Article  ADS  Google Scholar 

  15. Lerosey, G., De Rosny, J., Tourin, A. & Fink, M. Focusing beyond the diffraction limit with far-field time reversal. Science 315, 1120–1122 (2007).

    Article  ADS  Google Scholar 

  16. Katz, O., Small, E., Bromberg, Y. & Silberberg, Y. Focusing and compression of ultrashort pulses through scattering media. Nature Photon. 5, 372–377 (2011).

    Article  ADS  Google Scholar 

  17. Vellekoop, I. M., van Putten, E. G., Lagendijk, A. & Mosk, A. P. Demixing light paths inside disordered metamaterials. Opt. Express 16, 67–80 (2008).

    Article  ADS  Google Scholar 

  18. Hsieh, C. L., Pu, Y., Grange, R., Laporte, G. & Psaltis, D. Imaging through turbid layers by scanning the phase conjugated second harmonic radiation from a nanoparticle. Opt. Express 18, 20723–20731 (2010).

    Article  ADS  Google Scholar 

  19. Cui, M. & Yang, C. Implementation of a digital optical phase conjugation system and its application to study the robustness of turbidity suppression by phase conjugation. Opt. Express 18, 3444–3455 (2010).

    Article  ADS  Google Scholar 

  20. Cui, M., McDowell, E. J. & Yang, C. H. Observation of polarization-gate based reconstruction quality improvement during the process of turbidity suppression by optical phase conjugation. Appl. Phys. Lett. 95, 123702 (2009).

    Article  ADS  Google Scholar 

  21. Cui, M., McDowell, E. J. & Yang, C. An in vivo study of turbidity suppression by optical phase conjugation (TSOPC) on rabbit ear. Opt. Express 18, 25–30 (2010).

    Article  ADS  Google Scholar 

  22. Xu, X. A., Liu, H. L. & Wang, L. V. Time-reversed ultrasonically encoded optical focusing into scattering media. Nature Photon. 5, 154–157 (2011).

    Article  ADS  Google Scholar 

  23. Wang, L. V. Multiscale photoacoustic microscopy and computed tomography. Nature Photon. 3, 503–509 (2009).

    Article  ADS  Google Scholar 

  24. Yaqoob, Z., Psaltis, D., Feld, M. S. & Yang, C. Optical phase conjugation for turbidity suppression in biological samples. Nature Photon. 2, 110–115 (2008).

    Article  ADS  Google Scholar 

  25. Feinberg, J., Heiman, D., Tanguay, A. R. & Hellwarth, R. W. Photorefractive effects and light-induced charge migration in barium-titanate. J. Appl. Phys. 51, 1297–1305 (1980).

    Article  ADS  Google Scholar 

  26. Vellekoop, I. M. Controlling the Propagation of Light in Disordered Scattering Media. PhD thesis, Univ. Twente (2008).

  27. Lai, P. X., Xu, X., Liu, H. L., Suzuki, Y. & Wang, L. H. V. Reflection-mode time-reversed ultrasonically encoded optical focusing into turbid media. J. Biomed. Opt. 16, 080505 (2011).

    Article  ADS  Google Scholar 

  28. Wang, Y. M., Judkewitz, B., DiMarzio, C. A. & Yang, C. Deep-tissue focal fluorescence imaging with digitally time-reversed ultrasound-encoded light. Nature Commun. 3, 928 (2012).

    Article  ADS  Google Scholar 

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Acknowledgements

The authors thank C. Shank, Y.M. Wang and C. Yang for helpful discussions, T.-W. Chen for instructions on the micropipette puller and A. Hu for preparing the fixed rat brain slices. The research was supported by the Howard Hughes Medical Institute.

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Contributions

The experiment was designed and implemented by M.C. The fluorescence pattern was created by K.S. The scattering coefficient and the speckle correlation were measured by R.F. All authors contribute to the data analysis and preparation of the manuscript.

Corresponding author

Correspondence to Meng Cui.

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The authors declare no competing financial interests.

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Si, K., Fiolka, R. & Cui, M. Fluorescence imaging beyond the ballistic regime by ultrasound-pulse-guided digital phase conjugation. Nature Photon 6, 657–661 (2012). https://doi.org/10.1038/nphoton.2012.205

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