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Activation of mechanoluminescent nanotransducers by focused ultrasound enables light delivery to deep-seated tissue in vivo

Nature Protocols volume 18, pages 3787–3820 (2023)Cite this article

Subjects

Abstract

Light is used extensively in biological and medical research for optogenetic neuromodulation, fluorescence imaging, photoactivatable gene editing and light-based therapies. The major challenge to the in vivo implementation of light-based methods in deep-seated structures of the brain or of internal organs is the limited penetration of photons in biological tissue. The presence of light scattering and absorption has resulted in the development of invasive techniques such as the implantation of optical fibers, the insertion of endoscopes and the surgical removal of overlying tissues to overcome light attenuation and deliver it deep into the body. However, these procedures are highly invasive and make it difficult to reposition and adjust the illuminated area in each animal. Here, we detail a noninvasive approach to deliver light (termed ‘deLight’) in deep tissue via systemically injected mechanoluminescent nanotransducers that can be gated by using focused ultrasound. This approach achieves localized light emission with sub-millimeter resolution and millisecond response times in any vascularized organ of living mice without requiring invasive implantation of light-emitting devices. For example, deLight enables optogenetic neuromodulation in live mice without a craniotomy or brain implants. deLight provides a generalized method for applications that require a light source in deep tissues in vivo, such as deep-brain fluorescence imaging and photoactivatable genome editing. The implementation of the entire protocol for an in vivo application takes ~1–2 weeks.

Key points

  • This protocol describes the synthesis and characterization of mechanoluminescent nanotransducers covering the entire visible spectrum, their characterization in response to ultrasound in tissue-mimicking phantoms and in artificial circulatory systems and their use in mouse behavioral and immunohistochemical assays.

  • Alternative deep-tissue light-delivery approaches include implanted optical fibers or micro light-emitting diodes, intracranially injected upconversion nanoparticles and wavefront-shaping methods based on spatial light modulators.

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Fig. 1: Principles of deLight and its application in vivo.
Fig. 2: Synthesis of bulk mechanoluminescent materials via solid-state reactions.
Fig. 3: Bright-field and afterglow images of as-synthesized bulk mechanoluminescent materials.
Fig. 4: Synthesis process of biocompatible MLNTs from bulk mechanoluminescent materials.
Fig. 5: Calibration of the FUS system and characterizations of MLNTs in a tissue-mimicking phantom.
Fig. 6: Characterizations of MLNTs in an artificial circulatory system.
Fig. 7: Application of deLight in vivo.
Fig. 8: Dissection and staining of brain sections to evaluate the efficacy of deLight.
Fig. 9: Representative results of in vivo sono-optogenetic neuromodulation with deLight.

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Data availability

The source data for Extended Data Figs. 3–8 can be downloaded from https://doi.org/10.6084/m9.figshare.23690961. The STL file of the holder can be found at https://doi.org/10.6084/m9.figshare.23691309. A representative tiff file of the time series mechanoluminescence emission can be found at https://doi.org/10.6084/m9.figshare.23691312. Further data are available from the corresponding author upon request. Characterized and quality-controlled MLNTs are available to other research laboratories upon request.

Code availability

The custom LabVIEW and MATLAB codes used in this protocol are available at https://github.com/ShanJiang1233/deLight and are archived in Zenodo at https://doi.org/10.5281/zenodo.8162191.

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Acknowledgements

G.H. acknowledges two awards from the NIH (5R00AG056636-04 and 1R34NS127103-01), an NSF CAREER award (2045120), an NSF EAGER award (2217582), a Rita Allen Foundation Scholars Award, a Beckman Technology Development Grant, a grant from the Focused Ultrasound Foundation, a gift from the Spinal Muscular Atrophy (SMA) Foundation, a gift from the Pinetops Foundation, two seed grants from the Wu Tsai Neurosciences Institute and a seed grant from the Bio-X Initiative of Stanford University. X.W. acknowledges support from a Stanford Graduate Fellowship. N.J.R. acknowledges support from the NSF Graduate Research Fellowships Program (GRFP) and a Stanford Bio-X fellowship. Some schematics were created with BioRender.com.

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Author notes

  1. These authors contributed equally: Shan Jiang, Xiang Wu, Fan Yang.

Authors and Affiliations

  1. Department of Materials Science and Engineering, Stanford University, Stanford, CA, USA

    Shan Jiang, Xiang Wu, Fan Yang & Guosong Hong

  2. Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA

    Shan Jiang, Xiang Wu, Fan Yang, Nicholas J. Rommelfanger & Guosong Hong

  3. Department of Applied Physics, Stanford University, Stanford, CA, USA

    Nicholas J. Rommelfanger

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Contributions

S.J., X.W., F.Y., N.J.R. and G.H. contributed ideas and designed research. S.J. and F.Y. synthesized MLNTs. S.J., X.W., F.Y. and N.J.R. characterized MLNTs in PDMS phantoms and artificial circulatory systems under FUS. S.J. and X.W. performed the application of the deLight system for noninvasive sono-optogenetic neuromodulation in live mice. S.J., X.W., F.Y., N.J.R. and G.H. wrote the paper.

Corresponding author

Correspondence to Guosong Hong.

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The authors declare no competing interests.

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Nature Protocols thanks Hao Shen, Seok-Hyun Yun and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Hong, G. Science 369, 638 (2020): https://doi.org/10.1126/science.abd3636

Wang, W. et al. J. Am. Chem. Soc. 145, 1097–1107 (2023): https://doi.org/10.1021/jacs.2c10666

Yang, F. et al. J. Am. Chem. Soc. 144, 18406–18418 (2022): https://doi.org/10.1021/jacs.2c06724

Yang, F. et al. Sci. Adv. 8, eabo6743 (2022): https://doi.org/10.1126/sciadv.abo6743

Extended data

Extended Data Fig. 1

Representative TEM images of colloidal MLNTs of Sr2MgSi2O7:Eu,Dy, ZnS:Cu,Al, ZnS:Mn and CaTiO3:Pr. Figure reproduced with permission from ref. 22, ACS.

Extended Data Fig. 2 Peak pressure measured by the hydrophone (black) plotted against the percent amplitude of the FUS system.

Linear fitting (black) yields an empirical formula: P = 0.1A + 0.2, where P is the pressure in mega pascals and A is the amplitude in percent. Figure reproduced from ref. 20. under the Creative Commons license CC BY 4.0.

Extended Data Fig. 3

Pressure mapping of the 1.5-MHz FUS transducer in the x-y plane (at z0 = 0) (a) and in the x-z plane (at y0 = 0) (b).

Extended Data Fig. 4

Normalized mechanoluminescence spectra of MLNTs composed of Sr2MgSi2O7:Eu,Dy (a), ZnS:Cu,Al (b), ZnS:Mn (c) and CaTiO3:Pr (d). Figure adapted with permission from ref. 22, ACS.

Extended Data Fig. 5

An example mechanoluminescence image (a) and its corresponding emission line profile fitted to a Gaussian function (b) to obtain the full-width-at-half-maximum (FWHM).

Extended Data Fig. 6

Representative rechargeability curves of MLNTs composed of Sr2MgSi2O7:Eu,Dy (a), ZnS:Cu,Al (b), ZnS:Mn (c) and CaTiO3:Pr (d). Figure reproduced with permission from ref. 22, ACS.

Extended Data Fig. 7 Temporal kinetics of FUS-induced light emission from colloidal solutions of MLNTs.

a−d, Temporal kinetics of MLNT solutions composed of Sr2MgSi2O7:Eu,Dy (a), ZnS:Cu,Al (b), ZnS:Mn (c) and CaTiO3:Pr (d) upon 10-, 20- and 50-ms FUS pulses in the artificial circulatory system with an imaging frame rate of 200 Hz. e−h, Onset and offset times of mechanoluminescence emission measured from a–d. All data are presented as the mean ± s.d. of 10 independent measurements. ML, mechanoluminescence. Figure reproduced with permission from ref. 22, ACS.

Extended Data Fig. 8

Emission intensity from mechanoluminescent fluids composed of Sr2MgSi2O7:Eu,Dy (a), ZnS:Cu,Al (b), ZnS:Mn (c) and CaTiO3:Pr (d) under varying FUS pressure. Figure reproduced with permission from ref. 22, ACS.

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Jiang, S., Wu, X., Yang, F. et al. Activation of mechanoluminescent nanotransducers by focused ultrasound enables light delivery to deep-seated tissue in vivo. Nat Protoc 18, 3787–3820 (2023). https://doi.org/10.1038/s41596-023-00895-8

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