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Fluorescence imaging through dynamic scattering media with speckle-encoded ultrasound-modulated light correlation

Nature Photonics volume 14, pages 511–516 (2020)Cite this article

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Abstract

Fluorescence imaging is indispensable to biomedical research, and yet it remains challenging to image through dynamic scattering samples. Techniques that combine ultrasound and light as exemplified by ultrasound-assisted wavefront shaping have enabled fluorescence imaging through scattering media. However, the translation of these techniques into in vivo applications has been hindered by the lack of high-speed solutions to counter the fast speckle decorrelation of dynamic tissue. Here, we report an ultrasound-enabled optical imaging method that instead leverages the dynamic nature to perform imaging. The method utilizes the correlation between the dynamic speckle-encoded fluorescence and ultrasound-modulated light signal that originate from the same location within a sample. We image fluorescent targets with an improved resolution of ≤75 µm (versus a resolution of 1.3 mm with direct optical imaging) within a scattering medium with 17 ms decorrelation time. This new imaging modality paves the way for fluorescence imaging in highly scattering tissue in vivo.

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Fig. 1: Principle of FLUX.
Fig. 2: Imaging a fluorescent target inside a dynamic scattering sample and characterizing the system performance.
Fig. 3: Imaging multiple fluorescent targets inside a dynamic scattering sample with a speckle decorrelation time of 17 ms.

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Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding authors upon reasonable request.

Code availability

The code that supports the plots within this paper and other findings of this study is available from the corresponding authors upon reasonable request.

References

  1. Ntziachristos, V. Going deeper than microscopy: the optical imaging frontier in biology. Nat. Methods 7, 603–614 (2010).

    Google Scholar 

  2. Bertolotti, J. et al. Non-invasive imaging through opaque scattering layers. Nature 491, 232–234 (2012).

    ADS  Google Scholar 

  3. Katz, O., Heidmann, P., Fink, M. & Gigan, S. Non-invasive single-shot imaging through scattering layers and around corners via speckle correlations. Nat. Photon. 8, 784–790 (2014).

    ADS  Google Scholar 

  4. Katz, O., Small, E. & Silberberg, Y. Looking around corners and through thin turbid layers in real time with scattered incoherent light. Nat. Photon. 6, 549–553 (2012).

    ADS  Google Scholar 

  5. Papadopoulos, I. N., Jouhanneau, J. S., Poulet, J. F. A. & Judkewitz, B. Scattering compensation by focus scanning holographic aberration probing (F-SHARP). Nat. Photon. 11, 116–123 (2017).

    ADS  Google Scholar 

  6. Yoon, S. et al. Deep optical imaging within complex scattering media. Nat. Rev. Phys. 2, 141–158 (2020).

    Google Scholar 

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

    ADS  Google Scholar 

  8. Popoff, S. M. et al. Measuring the transmission matrix in optics: an approach to the study and control of light propagation in disordered media. Phys. Rev. Lett. 104, 100601 (2010).

    ADS  Google Scholar 

  9. Mosk, A. P., Lagendijk, A., Lerosey, G. & Fink, M. Controlling waves in space and time for imaging and focusing in complex media. Nat. Photon. 6, 283–292 (2012).

    ADS  Google Scholar 

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

    ADS  Google Scholar 

  11. Rotter, S. & Gigan, S. Light fields in complex media: mesoscopic scattering meets wave control. Rev. Mod. Phys. 89, 015005 (2017).

    ADS  Google Scholar 

  12. Yu, H. et al. Recent advances in wavefront shaping techniques for biomedical applications. Curr. Appl. Phys. 15, 632–641 (2015).

    ADS  Google Scholar 

  13. Park, J. H., Yu, Z., Lee, K. R., Lai, P. & Park, Y. K. Perspective: wavefront shaping techniques for controlling multiple light scattering in biological tissues: toward in vivo applications. APL Photon. 3, 100901 (2018).

    ADS  Google Scholar 

  14. Jang, M. et al. Relation between speckle decorrelation and optical phase conjugation (OPC)-based turbidity suppression through dynamic scattering media: a study on in vivo mouse skin. Biomed. Opt. Express 6, 72–85 (2015).

    Google Scholar 

  15. Qureshi, M. M. et al. In vivo study of optical speckle decorrelation time across depths in the mouse brain. Biomed. Opt. Express 8, 4855–4864 (2017).

    Google Scholar 

  16. Liu, Y. et al. Optical focusing deep inside dynamic scattering media with near-infrared time-reversed ultrasonically encoded (TRUE) light. Nat. Commun. 6, 5904 (2015).

    ADS  Google Scholar 

  17. Akbulut, D., Huisman, T. J., van Putten, E. G., Vos, W. L. & Mosk, A. P. Focusing light through random photonic media by binary amplitude modulation. Opt. Express 19, 4017–4029 (2011).

    ADS  Google Scholar 

  18. Cui, M. A high speed wavefront determination method based on spatial frequency modulations for focusing light through random scattering media. Opt. Express 19, 2989–2995 (2011).

    ADS  Google Scholar 

  19. Wang, D. et al. Focusing through dynamic tissue with millisecond digital optical phase conjugation. Optica 2, 728–735 (2015).

    ADS  Google Scholar 

  20. Conkey, D. B., Caravaca-Aguirre, A. M. & Piestun, R. High-speed scattering medium characterization with application to focusing light through turbid media. Opt. Express 20, 1733–1740 (2012).

    ADS  Google Scholar 

  21. Stockbridge, C. et al. Focusing through dynamic scattering media. Opt. Express 20, 15086–15092 (2012).

    ADS  Google Scholar 

  22. Kim, D. et al. Toward a miniature endomicroscope: pixelation-free and diffraction-limited imaging through a fiber bundle. Opt. Lett. 39, 1921–1924 (2014).

    ADS  Google Scholar 

  23. Ma, C., Zhou, F., Liu, Y. & Wang, L. V. Single-exposure optical focusing inside scattering media using binarized time-reversed adapted perturbation. Optica 2, 869–876 (2015).

    ADS  Google Scholar 

  24. Liu, Y., Ma, C., Shen, Y. & Wang, L. V. Bit-efficient, sub-millisecond wavefront measurement using a lock-in camera for time-reversal based optical focusing inside scattering media. Opt. Lett. 41, 1321–1324 (2016).

    ADS  Google Scholar 

  25. Blochet, B., Bourdieu, L. & Gigan, S. Focusing light through dynamical samples using fast continuous wavefront optimization. Opt. Lett. 42, 4994–4997 (2017).

    ADS  Google Scholar 

  26. Liu, Y., Ma, C., Shen, Y., Shi, J. & Wang, L. V. Focusing light inside dynamic scattering media with millisecond digital optical phase conjugation. Optica 4, 280–288 (2017).

    ADS  Google Scholar 

  27. Tzang, O. et al. Wavefront shaping in complex media with a 350 kHz modulator via a 1D-to-2D transform. Nat. Photon. 13, 788–793 (2019).

    ADS  Google Scholar 

  28. Xia, M., Li, D., Wang, L. & Wang, D. Fast optical wavefront engineering for controlling light propagation in dynamic turbid media. J. Innov. Opt. Health Sci. 12, 1930007 (2019).

    Google Scholar 

  29. Wei, X. et al. Real-time frequency-encoded spatiotemporal focusing through scattering media using a programmable 2D ultrafine optical frequency comb. Sci. Adv. 6, eaay1192 (2020).

    ADS  Google Scholar 

  30. Wang, L. V. & Hu, S. Photoacoustic tomography: in vivo imaging from organelles to organs. Science 335, 1458–1462 (2012).

    ADS  Google Scholar 

  31. Gateau, J., Chaigne, T., Katz, O., Gigan, S. & Bossy, E. Improving visibility in photoacoustic imaging using dynamic speckle illumination. Opt. Lett. 38, 5188–5191 (2013).

    ADS  Google Scholar 

  32. Chaigne, T. et al. Super-resolution photoacoustic fluctuation imaging with multiple speckle illumination. Optica 3, 54–57 (2016).

    ADS  Google Scholar 

  33. Chaigne, T., Arnal, B., Vilov, S., Bossy, E. & Katz, O. Super-resolution photoacoustic imaging via flow-induced absorption fluctuations. Optica 4, 1397–1404 (2017).

    ADS  Google Scholar 

  34. Lichtman, J. W. & Conchello, J.-A. Fluorescence microscopy. Nat. Methods 2, 910–919 (2005).

    Google Scholar 

  35. Kobayashi, M., Mizumoto, T., Shibuya, Y., Enomoto, M. & Takeda, M. Fluorescence tomography in turbid media based on acousto-optic modulation imaging. Appl. Phys. Lett. 89, 181102 (2006).

    ADS  Google Scholar 

  36. Huynh, N. T., Hayes-Gill, B. R., Zhang, F. & Morgan, S. P. Ultrasound modulated imaging of luminescence generated within a scattering medium. J. Biomed. Opt. 18, 20505 (2013).

    Google Scholar 

  37. Liu, Y., Feshitan, J. A., Wei, M.-Y., Borden, M. A. & Yuan, B. Ultrasound-modulated fluorescence based on fluorescent microbubbles. J. Biomed. Opt. 19, 85005 (2014).

    Google Scholar 

  38. Zhang, Q., Morgan, S. P. & Mather, M. L. Nanoscale ultrasound-switchable FRET-based liposomes for near-infrared fluorescence imaging in optically turbid media. Small 13, 1602895 (2017).

    Google Scholar 

  39. Yuan, B., Uchiyama, S., Liu, Y., Nguyen, K. T. & Alexandrakis, G. High-resolution imaging in a deep turbid medium based on an ultrasound-switchable fluorescence technique. Appl. Phys. Lett. 101, 033703 (2012).

    ADS  Google Scholar 

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

    ADS  Google Scholar 

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

    ADS  Google Scholar 

  42. Si, K., Fiolka, R. & Cui, M. Fluorescence imaging beyond the ballistic regime by ultrasound pulse guided digital phase conjugation. Nat. Photon. 6, 657–661 (2012).

    ADS  Google Scholar 

  43. Horstmeyer, R., Ruan, H. & Yang, C. Guidestar-assisted wavefront-shaping methods for focusing light into biological tissue. Nat. Photon. 9, 563–571 (2015).

    ADS  Google Scholar 

  44. Blochet, B., Joaquina, K., Blum, L., Bourdieu, L. & Gigan, S. Enhanced stability of the focus obtained by wavefront optimization in dynamical scattering media. Optica 6, 1554–1561 (2019).

    ADS  Google Scholar 

  45. Resink, S. G., Boccara, A. C. & Steenbergen, W. State-of-the art of acousto-optic sensing and imaging of turbid media. J. Biomed. Opt. 17, 40901 (2012).

    Google Scholar 

  46. Elson, D. S., Li, R., Dunsby, C., Eckersley, R. & Tang, M.-X. Ultrasound-mediated optical tomography: a review of current methods. Interface Focus 1, 632–648 (2011).

    Google Scholar 

  47. Gunther, J. & Andersson-Engels, S. Review of current methods of acousto-optical tomography for biomedical applications. Front. Optoelectron. 10, 211–238 (2017).

    Google Scholar 

  48. Ruan, H., Mather, M. L. & Morgan, S. P. Pulsed ultrasound modulated optical tomography utilizing the harmonic response of lock-in detection. Appl. Opt. 52, 4755–4762 (2013).

    ADS  Google Scholar 

  49. Dertinger, T., Colyera, R., Iyer, G., Weiss, S. & Enderlein, J. Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI). Proc. Natl Acad. Sci. USA 106, 22287–22292 (2009).

    ADS  Google Scholar 

  50. Doktofsky, D., Rosenfeld, M. & Katz, O. Acousto optic imaging beyond the acoustic diffraction limit using speckle decorrelation. Commun. Phys. 3, 5 (2020).

    Google Scholar 

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Acknowledgements

This work was supported by the Kernel–Brain Research and Technologies fund (FS 13520230) and the Rosen Bioengineering Center Endowment Fund (9900050).

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  1. These authors contributed equally: Haowen Ruan, Yan Liu.

Authors and Affiliations

  1. Department of Electrical Engineering, California Institute of Technology, Pasadena, CA, USA

    Haowen Ruan, Yan Liu, Jian Xu, Yujia Huang & Changhuei Yang

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  1. Haowen Ruan
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Contributions

H.R. conceived the idea. H.R., Y.L. and C.Y. developed the idea and designed the experiments. Y.L. and H.R. developed the experimental protocol and set-up. Y.L. constructed the samples and conducted the imaging experiments. H.R. and Y.L. analysed the data. C.Y., J.X., H.R., Y.L. and Y.H. conducted the theoretical analysis. All authors contributed to the preparation of the manuscript.

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Correspondence to Haowen Ruan, Yan Liu or Changhuei Yang.

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Supplementary Figs. 1–5 and Notes 1–4.

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Ruan, H., Liu, Y., Xu, J. et al. Fluorescence imaging through dynamic scattering media with speckle-encoded ultrasound-modulated light correlation. Nat. Photonics 14, 511–516 (2020). https://doi.org/10.1038/s41566-020-0630-0

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