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Deep laser microscopy using optical clearing by ultrasound-induced gas bubbles

Nature Photonics volume 16, pages 762–768 (2022)Cite this article

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Abstract

Although laser scanning microscopy is a pivotal imaging tool in biomedical research, optical scattering from tissue limits the depth of the imaging. To overcome this limitation, we propose a scheme called ultrasound-induced optical clearing microscopy, which makes use of temporary, localized optical clearing based on ultrasound-induced gas bubbles. In this method, bubbles are generated by high-intensity pulsed ultrasound at a desired depth and subsequently maintained by low-intensity continuous ultrasound during imaging. As a result, optical scattering and unwanted changes in the propagation direction of the incident photons are minimized in the bubble cloud, and thus the laser can be tightly focused at a deeper imaging plane. Through phantom and ex vivo experiments, we demonstrate that ultrasound-induced optical clearing microscopy is capable of increasing the imaging depth by a factor of six or more, while the resolution is similar to that of conventional laser scanning microscopy.

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Fig. 1: Principle of US-OCM for deep-tissue imaging.
Fig. 2: Light-intensity distribution at 350 and 450 μm depths in the tissue-mimicking phantom before and after bubble generation.
Fig. 3: Evaluation of the imaging performance of US-OCM.
Fig. 4: 3D images of 1 μm fluorescent beads inside the tissue-mimicking phantom.

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Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Code availability

The codes for the simulation are available from the corresponding authors upon reasonable request.

References

  1. Pawley, J. B. (ed.) Handbook of Biological Confocal Microscopy 3rd edn (Springer, 2006); https://doi.org/10.1007/978-0-387-45524-2

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

    Article  Google Scholar 

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

    Article  Google Scholar 

  4. Benninger, R. K. P. & Piston, D. W. Two-photon excitation microscopy for the study of living cells and tissues. Curr. Protoc. Cell Biol. 59, 4.11.1–4.11.24 (2013).

    Article  Google Scholar 

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

    Article  ADS  Google Scholar 

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

    Article  ADS  Google Scholar 

  7. Jang, M. et al. Deep tissue space-gated microscopy via acousto-optic interaction. Nat. Commun. 11, 710 (2020).

    Article  ADS  Google Scholar 

  8. Omar, M., Aguirre, J. & Ntziachristos, V. Optoacoustic mesoscopy for biomedicine. Nat. Biomed. Eng. 3, 354–370 (2019).

    Article  Google Scholar 

  9. Zhang, Z. et al. Imaging volumetric dynamics at high speed in mouse and zebrafish brain with confocal light field microscopy. Nat. Biotechnol. 39, 74–83 (2021).

    Article  Google Scholar 

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

    Article  ADS  Google Scholar 

  11. 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).

    Article  ADS  Google Scholar 

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

    Article  ADS  Google Scholar 

  13. Ruan, H., Liu, Y., Xu, J., Huang, Y. & Yang, C. Fluorescence imaging through dynamic scattering media with speckle-encoded ultrasound-modulated light correlation. Nat. Photonics 14, 511–516 (2020).

    Article  Google Scholar 

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

    Article  ADS  Google Scholar 

  15. Zhu, X. et al. Ultrafast optical clearing method for three-dimensional imaging with cellular resolution. Proc. Natl Acad. Sci. USA 116, 11480–11489 (2019).

    Article  ADS  Google Scholar 

  16. Costantini, I., Cicchi, R., Silvestri, L., Vanzi, F. & Pavone, F. S. In-vivo and ex-vivo optical clearing methods for biological tissues: review. Biomed. Opt. Express 10, 5251–5267 (2019).

    Article  Google Scholar 

  17. Yu, T., Zhu, J., Li, D. & Zhu, D. Physical and chemical mechanisms of tissue optical clearing. iScience 24, 102178 (2021).

    Article  ADS  Google Scholar 

  18. Jin, Y. L., Chen, J. Y., Xu, L. & Wang, P. N. Refractive index measurement for biomaterial samples by total internal reflection. Phys. Med. Biol. 51, N371–N379 (2006).

    Article  Google Scholar 

  19. Hsieh, Z.-H., Fan, C.-H., Ho, Y.-J., Li, M.-L. & Yeh, C.-K. Improvement of light penetration in biological tissue using an ultrasound-induced heating tunnel. Sci Rep. 10, 17406 (2020).

    Article  ADS  Google Scholar 

  20. Laufer, J., Simpson, R., Kohl, M., Essenpreis, M. & Cope, M. Effect of temperature on the optical properties of ex vivo human dermis and subdermis. Phys. Med. Biol. 43, 2479–2489 (1998).

    Article  Google Scholar 

  21. Su, Y. et al. Measurements of the thermal coefficient of optical attenuation at different depth regions of in vivo human skins using optical coherence tomography: a pilot study. Biomed. Opt. Express 6, 500–513 (2015).

    Article  Google Scholar 

  22. Shehata, I. A. Treatment with high intensity focused ultrasound: secrets revealed. Eur. J. Radiol. 81, 534–541 (2012).

    Article  Google Scholar 

  23. Ilovitsh, T. et al. Enhanced microbubble contrast agent oscillation following 250 kHz insonation. Sci Rep. 8, 16347 (2018).

    Article  ADS  Google Scholar 

  24. Maxwell, A. D., Cain, C. A., Hall, T. L., Fowlkes, J. B. & Xu, Z. Probability of cavitation for single ultrasound pulses applied to tissues and tissue-mimicking materials. Ultrasound Med. Biol. 39, 449–465 (2013).

    Article  Google Scholar 

  25. Vlaisavljevich, E. et al. Effects of tissue stiffness, ultrasound frequency, and pressure on histotripsy-induced cavitation bubble behavior. Phys. Med. Biol. 60, 2271–2292 (2015).

    Article  Google Scholar 

  26. Hoogenboom, M. et al. Mechanical high-intensity focused ultrasound destruction of soft tissue: working mechanisms and physiologic effects. Ultrasound Med. Biol. 41, 1500–1517 (2015).

    Article  Google Scholar 

  27. Lin, K.-W. et al. Histotripsy beyond the intrinsic cavitation threshold using very short ultrasound pulses: microtripsy. IEEE Trans. Ultrason. Ferroelectr. Freq. Control 61, 251–0885 (2014).

    Article  Google Scholar 

  28. Şen, T., Tüfekçioğlu, O. & Koza, Y. Mechanical index. Anatol. J. Cardiol. 15, 334–336 (2015).

    Article  Google Scholar 

  29. Marti, D., Aasbjerg, R. N., Andersen, P. E. & Hansen, A. K. MCmatlab: an open-source, user-friendly, MATLAB-integrated three-dimensional Monte Carlo light transport solver with heat diffusion and tissue damage. J. Biomed. Opt. 23, 121622 (2018).

    Article  ADS  Google Scholar 

  30. Kim, O. A., Ohmae, S. & Medina, J. F. A cerebello-olivary signal for negative prediction error is sufficient to cause extinction of associative motor learning. Nat. Neurosci. 23, 1550–1554 (2020).

    Article  Google Scholar 

  31. McLaughlan, J., Rivens, I., Leighton, T. & ter Haar, G. A study of bubble activity generated in ex vivo tissue by high intensity focused ultrasound. Ultrasound Med. Biol. 36, 1327–1344 (2010).

    Article  Google Scholar 

  32. Nagarajan, V. K. & Yu, B. Monitoring of tissue optical properties during thermal coagulation of ex vivo tissues. Lasers Surg. Med. 48, 686–694 (2016).

    Article  Google Scholar 

  33. Kim, H. & Chang, J. H. Increased light penetration due to ultrasound-induced air bubbles in optical scattering media. Sci Rep. 7, 16105 (2017).

    Article  ADS  Google Scholar 

  34. Vaezy, S. et al. Real-time visualization of high-intensity focused ultrasound treatment using ultrasound imaging. Ultrasound Med. Biol. 27, 33–42 (2001).

    Article  Google Scholar 

  35. Kim, J., Kim, H. & Chang, J. H. Endoscopic probe for ultrasound-assisted photodynamic therapy of deep-lying tissue. IEEE Access 8, 179745–179753 (2020).

    Article  Google Scholar 

  36. Prentice, P., Cuschieri, A., Dholakia, K., Prausnitz, M. & Campbell, P. Membrane disruption by optically controlled microbubble cavitation. Nat. Phys. 1, 107–110 (2005).

    Article  Google Scholar 

  37. Fan, Q., Hu, W. & Ohta, A. T. Laser-induced microbubble poration of localized single cells. Lab Chip 14, 1572–1578 (2014).

    Article  Google Scholar 

  38. Mao, M., Montgomery, J. M., Kubke, M. F. & Thorne, P. R. The structural development of the mouse dorsal cochlear nucleus. J. Assoc. Res. Otolaryngol. 16, 473–486 (2015).

    Article  Google Scholar 

  39. Tario, J. D. Jr et al. Optimized staining and proliferation modeling methods for cell division monitoring using cell tracking dyes. J. Vis. Exp. e4287 (2012); https://doi.org/10.3791/4287

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Acknowledgements

This work was supported by the Samsung Research Funding Center of Samsung Electronics under project number SRFC-IT1702-03 (J.H.C. and J.Y.W.).

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Authors and Affiliations

  1. Department of Electrical Engineering and Computer Science, DGIST (Daegu Gyeongbuk Institute of Science and Technology), Daegu, South Korea

    Haemin Kim, Sangyeon Youn, Jinwoo Kim, Moonhwan Lee, Jae Youn Hwang & Jin Ho Chang

  2. Department of Electronic Engineering, Sogang University, Seoul, South Korea

    Sunghun Park

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Contributions

J.H.C. conceived the idea, and J.H.C. and J.Y.H. developed the idea and designed the project. H.K., J.Y.H. and J.H.C. developed the experimental protocol. S.Y., J.K. and M.L. designed and built the US-OCM system. S.P. performed the ultrasound beam simulations and analysed the standing-wave properties. H.K. collected the experimental data, performed the Monte Carlo simulations and wrote the initial manuscript draft. J.H.C. and J.Y.H. revised the draft. All authors were involved in the analysis of the results and revision of the manuscript.

Corresponding authors

Correspondence to Jae Youn Hwang or Jin Ho Chang.

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Nature Photonics thanks the anonymous reviewers for their contribution to the peer review of this work.

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Supplementary Information

Supplementary description of the developed system, simulation and experimental results, discussion and Figs. 1–15.

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Kim, H., Youn, S., Kim, J. et al. Deep laser microscopy using optical clearing by ultrasound-induced gas bubbles. Nat. Photon. 16, 762–768 (2022). https://doi.org/10.1038/s41566-022-01068-x

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