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Achieving optical transparency in live animals with absorbing molecules | Science
Achieving optical transparency in live animals with absorbing molecules \| Science

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[Home](https://www.science.org/ "Home") [Science](/journal/science "Science") [Vol. 385, No. 6713](/toc/science/385/6713 "Vol. 385, No. 6713") Achieving optical transparency in live animals with absorbing molecules

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# Achieving optical transparency in live animals with absorbing molecules

[ZihaoOu](#con1) [https://orcid.org/0000-0003-2987-7423](https://orcid.org/0000-0003-2987-7423), [Yi-ShiouDuh](#con2) [https://orcid.org/0009-0008-3702-1610](https://orcid.org/0009-0008-3702-1610), \[...\], [Nicholas J.Rommelfanger](#con3) [https://orcid.org/0000-0002-1367-3939](https://orcid.org/0000-0002-1367-3939), [Carl H. C.Keck](#con4) [https://orcid.org/0000-0003-1944-4165](https://orcid.org/0000-0003-1944-4165), \[...\], [ShanJiang](#con5) [https://orcid.org/0000-0002-1567-2745](https://orcid.org/0000-0002-1567-2745), [KennethBrinson Jr](#con6) [https://orcid.org/0000-0002-1447-9169](https://orcid.org/0000-0002-1447-9169), [SuZhao](#con7) [https://orcid.org/0000-0001-5797-9190](https://orcid.org/0000-0001-5797-9190), [Elizabeth L.Schmidt](#con8) [https://orcid.org/0000-0002-7420-2997](https://orcid.org/0000-0002-7420-2997), [XiangWu](#con9) [https://orcid.org/0000-0001-8872-7738](https://orcid.org/0000-0001-8872-7738), \[...\], [FanYang](#con10) [https://orcid.org/0000-0001-6306-4501](https://orcid.org/0000-0001-6306-4501), [BettyCai](#con11), [HanCui](#con12) [https://orcid.org/0000-0001-6473-9232](https://orcid.org/0000-0001-6473-9232), [WeiQi](#con13), [ShifuWu](#con14), [AdarshTantry](#con15) [https://orcid.org/0000-0002-4211-5549](https://orcid.org/0000-0002-4211-5549), [RichardRoth](#con16) [https://orcid.org/0000-0002-6855-999X](https://orcid.org/0000-0002-6855-999X), [JunDing](#con17) [https://orcid.org/0000-0003-0690-1312](https://orcid.org/0000-0003-0690-1312), [XiaokeChen](#con18) [https://orcid.org/0000-0001-9032-9093](https://orcid.org/0000-0001-9032-9093), [Julia A.Kaltschmidt](#con19) [https://orcid.org/0000-0002-2893-1793](https://orcid.org/0000-0002-2893-1793), [Mark L.Brongersma](#con20) [https://orcid.org/0000-0003-1777-8970](https://orcid.org/0000-0003-1777-8970) [\[email protected\]](/cdn-cgi/l/email-protection#91f3e3fefff6f4e3e2fcf0d1e2e5f0fff7fee3f5bff4f5e4aef2f2acf6e4fee2fefff6f9d1e2e5f0fff7fee3f5bff4f5e4), and [GuosongHong](#con21) [https://orcid.org/0000-0002-8858-4471](https://orcid.org/0000-0002-8858-4471) [\[email protected\]](/cdn-cgi/l/email-protection#482a3a27262f2d3a3b2529083b3c29262e273a2c662d2c3d772b2b752f3d273b27262f20083b3c29262e273a2c662d2c3d)+18 authors+16 authors+11 authorsfewer [Authors Info & Affiliations](#tab-contributors)

Science

6 Sep 2024

Vol 385, Issue 6713

[DOI: 10.1126/science.adm6869](https://doi.org/10.1126/science.adm6869)

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- Contents

- [Editor’s summary](#editor-abstract)
- [Structured Abstract](#structured-abstract)
- [Abstract](#abstract)
- [Supplementary Materials](#supplementary-materials)
- [References and Notes](#bibliography)

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## Editor’s summary

Optical imaging of biological tissues is hindered by the scattering and, to a lesser extent, absorption of light that limits the penetration depth. Ou _et al_. addressed this problem through an approach that at first may seem counterintuitive: the introduction of highly absorbing molecules (see the Perspective by Rowlands and Gorecki). The authors show that the addition of common dye molecules that absorb in the near ultraviolet and blue regions improve optical transparency in nearby longer wavelengths. In essence, by causing sharp absorption in the blue region, the refractive index in the red part of the spectrum is increased without increasing absorption. The addition of tartrazine was able to make the skin of a live rodent temporarily transparent. —Marc S. Lavine

## Structured Abstract

### INTRODUCTION

A challenge in trying to image biological matter is that its complex structure causes opacity because of unwanted light scattering. This scattering results from refractive index mismatches among the components of biological tissues, limiting the penetration depth of optical imaging. The desire to see inside biological tissue and uncover the fundamental processes of life has spurred extensive research into deep-tissue optical imaging methods, such as two-photon microscopy, near-infrared-II fluorescence imaging, and optical tissue clearing. However, these methods either lack sufficient penetration depth and resolution or are unsuitable for living animals. Therefore, the ability to achieve optical transparency in live animals holds promise for transforming many optical imaging techniques.

### RATIONALE

We hypothesized that strongly absorbing molecules can achieve optical transparency in live biological tissues. By applying the Lorentz oscillator model for the dielectric properties of tissue components and absorbing molecules, we predicted that dye molecules with sharp absorption resonances in the near-ultraviolet spectrum (300 to 400 nm) and blue region of the visible spectrum (400 to 500 nm) are effective in raising the real part of the refractive index of the aqueous medium at longer wavelengths when dissolved in water, which is in agreement with the Kramers-Kronig relations. As a result, water-soluble dyes can effectively reduce the RI contrast between water and lipids, leading to optical transparency of live biological tissues.

### RESULTS

Following our theory, we found that an aqueous solution of a common food color approved by the US Food and Drug Administration, tartrazine, has the effect of reversibly making the skin, muscle, and connective tissues transparent in live rodents. We conducted experiments in both tissue-mimicking scattering hydrogels and ex vivo biological tissues. These tests confirmed the mechanism underlying our observations and showcased the achievable spatial resolution down to the micrometer level through millimeters of scattering medium once transparency is attained. By using absorbing dye molecules, we can transform the typically opaque abdomen of a live mouse into a transparent medium. This “transparent abdomen” allows for direct visualization of fluorescent protein–labeled enteric neurons, capturing their movements that mirror the underlying gut motility in live mice. This enabled us to generate time-evolving maps that depict mouse gut motility and the diversity of movement patterns. To demonstrate the generalizability of this approach, we also applied dye solutions topically to the scalp of a mouse head for visualizing cerebral blood vessels and to the mouse hindlimb for high-resolution microscopic imaging of muscle sarcomeres.

### CONCLUSION

Overall, we report the counterintuitive observation that strongly absorbing molecules can achieve optical transparency in live animals. The Lorentz oscillator model, which underlies this unusual observation, predicts that molecules with low resonance frequencies (long absorption wavelengths), sharp absorption peaks, and rich delocalized electrons are more effective candidates at raising the refractive index of the aqueous medium than are conventional optical clearing agents. Our approach also presents opportunities for visualizing the structure, activity, and functions of deep-seated tissues and organs without the need for surgical removal or the replacement of overlying tissues with transparent windows. Some limitations remain for this method, including reduced but not eliminated scattering owing to the challenge of matching refractive indices in heterogeneous tissues and achievable penetration depth depending on the diffusion of absorbing molecules.

![](/cms/10.1126/science.adm6869/asset/16de2ae5-74b6-4d4a-8792-b4fd7bcfbf2b/assets/images/large/science.adm6869-fa.jpg)

Achieving optical transparency in live mice with absorbing dye molecules.

Strongly absorbing molecules dissolved in water can modify the RI of the aqueous medium through the Kramers-Kronig relations to match that of lipids. This approach can render various samples transparent, including scattering phantoms, chicken breast tissue, and live mouse body for visualizing a wide range of deep-seated structures and activities. Scale bars, 5 mm. \[The schematic was prepared using BioRender.com\]

## Abstract

Optical imaging plays a central role in biology and medicine but is hindered by light scattering in live tissue. We report the counterintuitive observation that strongly absorbing molecules can achieve optical transparency in live animals. We explored the physics behind this observation and found that when strongly absorbing molecules dissolve in water, they can modify the refractive index of the aqueous medium through the Kramers-Kronig relations to match that of high-index tissue components such as lipids. We have demonstrated that our straightforward approach can reversibly render a live mouse body transparent to allow visualization of a wide range of deep-seated structures and activities. This work suggests that the search for high-performance optical clearing agents should focus on strongly absorbing molecules.

Related Perspective

[Turning tissues temporarily transparent](/doi/10.1126/science.adr7935)

By Christopher J. Rowlands, Jon Gorecki•Science•6 Sep 2024

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## Supplementary Materials

### The PDF file includes:

Supplementary Text

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References ( [_58_](#R58), [_59_](#R59))

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### Other Supplementary Material for this manuscript includes the following:

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#### Acknowledgments

Ellipsometry characterizations were performed at the Stanford Nano Shared Facilities (SNSF), supported by the National Science Foundation under award ECCS-1542152. Any opinions, findings, and conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. In vivo two-photon imaging experiments were performed at Stanford Wu Tsai Neuroscience Microscopy Service, with help from G. Wang. We gratefully acknowledge M. Goodman and A. Baghdasaryan at Stanford University for useful discussions and assistance.

**Funding:** This work was supported by National Institutes of Health grant 5R00AG056636 (G.H.); National Institutes of Health grant 1R34NS127103 (G.H.); National Institutes of Health grant R01NS126076 (G.H.); National Institutes of Health grant R01NS129834 (X.C.); National Institutes of Health grant R01DA059602 (X.C.); National Institutes of Health grant R01MH116904 (X.C.); National Institutes of Health grant R01DA045664 (X.C.); National Science Foundation CAREER award 2045120 (G.H.); National Science Foundation EAGER award 2217582 (G.H.); National Science Foundation Graduate Research Fellowships program 1656518 (N.J.R. and C.H.C.K.); Air Force Office of Scientific Research grant FA9550-21-1-0312 (M.L.B.); a Beckman Technology Development grant (J.A.K. and G.H.); a Rita Allen Foundation Scholars award (G.H.); the Focused Ultrasound Foundation (G.H.); the Spinal Muscular Atrophy Foundation (G.H.); the Pinetops Foundation (G.H.); the Bio-X Initiative of Stanford University (J.A.K. and G.H.); a Bio-X Graduate Student Fellowship (Y.-S.D. and N.J.R.); a Stanford Interdisciplinary Graduate Fellowship (H.C.); a Wu Tsai Neuroscience Institute seed grant (G.H.); the Wu Tsai Neuroscience Interdisciplinary Scholar program (Z.O.); the Wu Tsai Neuroscience NeuroTech Training program (C.H.C.K.); a Stanford Graduate Fellowship (X.W.); the Knight-Hennessy Scholarship program (K.B.); and the US Army Long Term Health Education and Training program (K.B.).

**Author contributions:** Conceptualization: Z.O., N.J.R., M.L.B., and G.H. Materials absorption characterization: Z.O. and B.C. Ellipsometry characterization: Z.O. Silica phantom characterization: Z.O., X.W., and F.Y. FDTD simulation: Y.-S.D. and M.L.B. Ex vivo tissue characterization: Z.O. and S.W. In vivo animal experiments: Z.O., C.H.C.K., S.J., K.B., S.Z., E.L.S., H.C., W.Q., A.T., R.R., J.D., X.C., J.A.K., and G.H. Supervision: J.A.K., M.L.B., and G.H. Writing – original draft: Z.O., Y.-S.D., N.J.R., M.L.B., G.H. Writing – review and editing: Z.O., Y.-S.D., N.J.R., C.H.C.K., E.L.S., A.T., R.R., J.D., X.C., J.A.K., M.L.B., and G.H.

**Competing interests:** G.H., Z.O., N.J.R., S.J., M.L.B., Y.-S.D., K.B., and S.Z. are inventors on patent application (WO2023122534A1) submitted by Stanford University that covers the principles of achieving optical transparency by applying the Kramers-Kronig relations.

**Data and materials availability:** All data are available in the manuscript or in the supplementary materials. Aqueous solutions and hydrogels containing dye molecules are available from G.H. under a materials transfer agreement with Stanford University. The code for all simulations and data analyses are provided on Zenodo ( [_57_](#R57)).

**License information:** Copyright © 2024 the authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original US government works. [https://www.science.org/about/science-licenses-journal-article-reuse](https://www.science.org/about/science-licenses-journal-article-reuse)

### Authors

#### Affiliations

ZihaoOu [https://orcid.org/0000-0003-2987-7423](https://orcid.org/0000-0003-2987-7423)

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

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

Roles: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing - original draft, and Writing - review & editing.

[View all articles by this author](/authored-by/Ou/Zihao)

Yi-ShiouDuh [https://orcid.org/0009-0008-3702-1610](https://orcid.org/0009-0008-3702-1610)

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

Geballe Laboratory for Advanced Materials, Stanford University, Stanford, CA, USA.

Roles: Formal analysis, Methodology, and Software.

[View all articles by this author](/authored-by/Duh/Yi-Shiou)

Nicholas J.Rommelfanger [https://orcid.org/0000-0002-1367-3939](https://orcid.org/0000-0002-1367-3939)

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

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

Roles: Conceptualization, Software, Writing - original draft, and Writing - review & editing.

[View all articles by this author](/authored-by/Rommelfanger/Nicholas+J)

Carl H. C.Keck [https://orcid.org/0000-0003-1944-4165](https://orcid.org/0000-0003-1944-4165)

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

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

Roles: Investigation, Methodology, Resources, Validation, and Writing - review & editing.

[View all articles by this author](/authored-by/Keck/Carl+H+C)

ShanJiang [https://orcid.org/0000-0002-1567-2745](https://orcid.org/0000-0002-1567-2745)

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

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

Roles: Conceptualization, Investigation, Methodology, and Resources.

[View all articles by this author](/authored-by/Jiang/Shan)

KennethBrinson Jr [https://orcid.org/0000-0002-1447-9169](https://orcid.org/0000-0002-1447-9169)

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

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

Role: Investigation.

[View all articles by this author](/authored-by/Brinson/Kenneth/Jr)

SuZhao [https://orcid.org/0000-0001-5797-9190](https://orcid.org/0000-0001-5797-9190)

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

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

Roles: Investigation, Methodology, Resources, Validation, and Writing - review & editing.

[View all articles by this author](/authored-by/Zhao/Su)

Elizabeth L.Schmidt [https://orcid.org/0000-0002-7420-2997](https://orcid.org/0000-0002-7420-2997)

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

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

Roles: Investigation, Methodology, and Writing - review & editing.

[View all articles by this author](/authored-by/Schmidt/Elizabeth+L)

XiangWu [https://orcid.org/0000-0001-8872-7738](https://orcid.org/0000-0001-8872-7738)

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

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

Roles: Investigation and Methodology.

[View all articles by this author](/authored-by/Wu/Xiang)

FanYang [https://orcid.org/0000-0001-6306-4501](https://orcid.org/0000-0001-6306-4501)

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

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

Roles: Investigation, Methodology, Resources, and Validation.

[View all articles by this author](/authored-by/Yang/Fan)

BettyCai

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

Roles: Investigation and Writing - review & editing.

[View all articles by this author](/authored-by/Cai/Betty)

HanCui [https://orcid.org/0000-0001-6473-9232](https://orcid.org/0000-0001-6473-9232)

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

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

Roles: Investigation and Writing - original draft.

[View all articles by this author](/authored-by/Cui/Han)

WeiQi

Department of Biology, Stanford University, Stanford, CA, USA.

Role: Investigation.

[View all articles by this author](/authored-by/Qi/Wei)

ShifuWu

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

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

Role: Investigation.

[View all articles by this author](/authored-by/Wu/Shifu)

AdarshTantry [https://orcid.org/0000-0002-4211-5549](https://orcid.org/0000-0002-4211-5549)

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

Neurosciences IDP Graduate program, Stanford University, Stanford, CA.

Roles: Resources and Writing - review & editing.

[View all articles by this author](/authored-by/Tantry/Adarsh)

RichardRoth [https://orcid.org/0000-0002-6855-999X](https://orcid.org/0000-0002-6855-999X)

Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA.

Roles: Investigation, Resources, and Writing - review & editing.

[View all articles by this author](/authored-by/Roth/Richard)

JunDing [https://orcid.org/0000-0003-0690-1312](https://orcid.org/0000-0003-0690-1312)

Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA.

Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA.

Roles: Investigation, Resources, Supervision, Validation, Visualization, and Writing - review & editing.

[View all articles by this author](/authored-by/Ding/Jun)

XiaokeChen [https://orcid.org/0000-0001-9032-9093](https://orcid.org/0000-0001-9032-9093)

Department of Biology, Stanford University, Stanford, CA, USA.

Roles: Conceptualization, Funding acquisition, Project administration, and Supervision.

[View all articles by this author](/authored-by/Chen/Xiaoke)

Julia A.Kaltschmidt [https://orcid.org/0000-0002-2893-1793](https://orcid.org/0000-0002-2893-1793)

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

Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA.

Roles: Conceptualization, Funding acquisition, Resources, Supervision, and Writing - review & editing.

[View all articles by this author](/authored-by/Kaltschmidt/Julia+A)

Mark L.Brongersma[\*](#cor1) [https://orcid.org/0000-0003-1777-8970](https://orcid.org/0000-0003-1777-8970) [\[email protected\]](/cdn-cgi/l/email-protection#3153435e5f565443425c50714245505f575e43551f5455440e52520c56445e425e5f5659714245505f575e43551f545544)

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

Geballe Laboratory for Advanced Materials, Stanford University, Stanford, CA, USA.

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

Roles: Conceptualization, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, and Writing - review & editing.

[View all articles by this author](/authored-by/Brongersma/Mark+L)

GuosongHong[\*](#cor1) [https://orcid.org/0000-0002-8858-4471](https://orcid.org/0000-0002-8858-4471) [\[email protected\]](/cdn-cgi/l/email-protection#0567776a6b626077766864457671646b636a77612b6061703a66663862706a766a6b626d457671646b636a77612b606170)

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

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

Roles: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Software, Supervision, Validation, Visualization, Writing - original draft, and Writing - review & editing.

[View all articles by this author](/authored-by/Hong/Guosong)

#### Funding Information

[National Science Foundation](http://dx.doi.org/10.13039/100000001): CAREER award 2045120

[National Science Foundation](http://dx.doi.org/10.13039/100000001): EAGER award 2217582

[National Science Foundation](http://dx.doi.org/10.13039/100000001): Graduate Research Fellowships program 1656518

[National Science Foundation](http://dx.doi.org/10.13039/100000001): Graduate Research Fellowships program 1656518

[National Institutes of Health](http://dx.doi.org/10.13039/100000002): 5R00AG056636-04

[National Institutes of Health](http://dx.doi.org/10.13039/100000002): 1R34NS127103-01

[National Institutes of Health](http://dx.doi.org/10.13039/100000002): R01NS126076-01

[Air Force Office of Scientific Research](http://dx.doi.org/10.13039/100000181): FA9550-21-1-0312

Bio-X Graduate Student Fellowship

Wu Tsai Neuroscience NeuroTech Training program

[Rita Allen Foundation](http://dx.doi.org/10.13039/100001447)

[Focused Ultrasound Foundation](http://dx.doi.org/10.13039/100006599)

Pinetops Foundation

[Spinal Muscular Atrophy Foundation](http://dx.doi.org/10.13039/100000894)

Beckman Technology Development

Bio-X Initiative of Stanford University

Wu Tsai Neuroscience Institute seed grant

Beckman Technology Development

Bio-X Initiative of Stanford University

Stanford Graduate Fellowship

Bio-X Graduate Student Fellowship

Knight-Hennessy Scholarship program

U.S. Army Long Term Health Education and Training program

Wu Tsai Neuroscience Interdisciplinary Scholar program

Stanford Interdisciplinary Graduate Fellowship

#### Notes

\*

Corresponding author. Email: [\[email protected\]](/cdn-cgi/l/email-protection#2745554849404255544a4667545346494148554309424352) (M.L.B.); [\[email protected\]](/cdn-cgi/l/email-protection#1e796b716d717079765e6d6a7f7078716c7a307b7a6b) (G.H.)

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Turning tissues temporarily transparent, Science, **385**, 6713, (1046-1047), (2024). [/doi/10.1126/science.adr7935](/doi/10.1126/science.adr7935 "Abstract")
[Abstract](/doi/10.1126/science.adr7935)

2. - Jude Coleman,


Transparent mice made with light-absorbing dye reveal organs at work, Nature, (2024). [https://doi.org/10.1038/d41586-024-02887-4](https://doi.org/10.1038/d41586-024-02887-4 "Crossref")
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