Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Through-skull fluorescence imaging of the brain in a new near-infrared window

Nature Photonics volume 8, pages 723–730 (2014)Cite this article

Subjects

Abstract

To date, brain imaging has largely relied on X-ray computed tomography and magnetic resonance angiography, with their limited spatial resolution and long scanning times. Fluorescence-based brain imaging in the visible and traditional near-infrared regions (400–900 nm) is an alternative, but at present it requires craniotomy, cranial windows and skull-thinning techniques, and the penetration depth is limited to 1–2 mm due to light scattering. Here, we report through-scalp and through-skull fluorescence imaging of mouse cerebral vasculature without craniotomy, utilizing the intrinsic photoluminescence of single-walled carbon nanotubes in the 1.3–1.4 μm near-infrared window (NIR-IIa window). Reduced photon scattering in this spectral region allows fluorescence imaging to a depth of >2 mm in mouse brain with sub-10-μm resolution. An imaging rate of ∼5.3 frames per second allows for dynamic recording of blood perfusion in the cerebral vessels with sufficient temporal resolution, providing real-time assessment of a blood flow anomaly in a mouse middle cerebral artery occlusion stroke model.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Imaging in various NIR subregions.
Figure 2: In vivo mouse brain imaging with SWNT–IRDye800 in different NIR subregions.
Figure 3: Non-invasive, high-resolution NIR-IIa fluorescence imaging of mouse brain vasculature.
Figure 4: Dynamic NIR-IIa fluorescence imaging of mouse cerebral vasculature.

Similar content being viewed by others

References

  1. Go, A. S. et al. Heart disease and stroke statistics—2013 update—a report from the American Heart Association. Circulation 127, E6–E245 (2013).

    Google Scholar 

  2. Schramm, P. et al. Comparison of CT and CT angiography source images with diffusion-weighted imaging in patients with acute stroke within 6 hours after onset. Stroke 33, 2426–2432 (2002).

    Article  Google Scholar 

  3. Wright, S. N. et al. Digital reconstruction and morphometric analysis of human brain arterial vasculature from magnetic resonance angiography. Neuroimage 82, 170–181 (2013).

    Article  Google Scholar 

  4. Huang, C.-H. et al. High-resolution structural and functional assessments of cerebral microvasculature using 3D gas ΔR2*-mMRA. PloS ONE 8, e78186 (2013).

    Article  ADS  Google Scholar 

  5. Flohr, T. G. et al. First performance evaluation of a dual-source CT (DSCT) system. Eur. Radiol. 16, 256–268 (2006).

    Article  Google Scholar 

  6. Jacoby, C. et al. Dynamic changes in murine vessel geometry assessed by high-resolution magnetic resonance angiography: a 9.4 T study. J. Magn. Reson. Imaging 28, 637–645 (2008).

    Article  Google Scholar 

  7. Paulus, M. J., Gleason, S. S., Kennel, S. J., Hunsicker, P. R. & Johnson, D. K. High resolution X-ray computed tomography: an emerging tool for small animal cancer research. Neoplasia 2, 62–70 (2000).

    Article  Google Scholar 

  8. Horton, N. G. et al. In vivo three-photon microscopy of subcortical structures within an intact mouse brain. Nature Photon. 7, 205–209 (2013).

    Article  ADS  Google Scholar 

  9. Svoboda, K., Denk, W., Kleinfeld, D. & Tank, D. W. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385, 161–165 (1997).

    Article  ADS  Google Scholar 

  10. Drew, P. J. et al. Chronic optical access through a polished and reinforced thinned skull. Nature Methods 7, 981–984 (2010).

    Article  Google Scholar 

  11. Yang, G., Pan, F., Parkhurst, C. N., Grutzendler, J. & Gan, W.-B. Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nature Protoc. 5, 201–208 (2010).

    Article  Google Scholar 

  12. Martirosyan, N. L. et al. Use of in vivo near-infrared laser confocal endomicroscopy with indocyanine green to detect the boundary of infiltrative tumor. Laboratory investigation. J. Neurosurg. 115, 1131–1138 (2011).

    Article  Google Scholar 

  13. 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 

  14. Kobat, D., Horton, N. G. & Xu, C. In vivo two-photon microscopy to 1.6-mm depth in mouse cortex. J. Biomed. Opt. 16, 106014 (2011).

    Article  ADS  Google Scholar 

  15. Splinter, R. & Hooper, B. A. An Introduction to Biomedical Optics (Taylor & Francis, 2007).

    Google Scholar 

  16. Frangioni, J. V. In vivo near-infrared fluorescence imaging. Curr. Opin. Chem. Biol. 7, 626–634 (2003).

    Article  Google Scholar 

  17. Smith, A. M., Mancini, M. C. & Nie, S. Bioimaging: second window for in vivo imaging. Nature Nanotech. 4, 710–711 (2009).

    Article  ADS  Google Scholar 

  18. Yi, H. et al. M13 phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors. Nano Lett. 12, 1176–1183 (2012).

    Article  ADS  Google Scholar 

  19. Welsher, K. et al. A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice. Nature Nanotech. 4, 773–780 (2009).

    Article  ADS  Google Scholar 

  20. Welsher, K., Sherlock, S. P. & Dai, H. Deep-tissue anatomical imaging of mice using carbon nanotube fluorophores in the second near-infrared window. Proc. Natl Acad. Sci. USA 108, 8943–8948 (2011).

    Article  ADS  Google Scholar 

  21. Hong, G. et al. Three-dimensional imaging of single nanotube molecule endocytosis on plasmonic substrates. Nature Commun. 3, 700 (2012).

    Article  ADS  Google Scholar 

  22. Hong, G. et al. Multifunctional in vivo vascular imaging using near-infrared II fluorescence. Nature Med. 18, 1841–1846 (2012).

    Article  Google Scholar 

  23. Won, N. et al. Imaging depths of near-infrared quantum dots in first and second optical windows. Mol. Imaging 11, 338–352 (2012).

    Article  Google Scholar 

  24. Hong, G. et al. In vivo fluorescence imaging with Ag2S quantum dots in the second near-infrared region. Angew. Chem. Int. Ed. 51, 9818–9821 (2012).

    Article  Google Scholar 

  25. Naczynski, D. J. et al. Rare-earth-doped biological composites as in vivo shortwave infrared reporters. Nature Commun. 4, 2199 (2013).

    Article  ADS  Google Scholar 

  26. Tao, Z. et al. Biological imaging using nanoparticles of small organic molecules with fluorescence emission at wavelengths longer than 1000 nm. Angew. Chem. Int. Ed. 52, 13002–13006 (2013).

    Article  Google Scholar 

  27. Dong, B. et al. Facile synthesis of highly photoluminescent Ag2Se quantum dots as a new fluorescent probe in the second near-infrared window for in vivo imaging. Chem. Mater. 25, 2503–2509 (2013).

    Article  Google Scholar 

  28. Hong, G. et al. Near-infrared II fluorescence for imaging hindlimb vessel regeneration with dynamic tissue perfusion measurement. Circ. Cardiovasc. Imaging 7, 517–525 (2014).

    Article  Google Scholar 

  29. Hong, G. et al. Ultrafast fluorescence imaging in vivo with conjugated polymer fluorophores in the second near-infrared window. Nature Commun. 5, 4206 10.1038/ncomms5206(2014).

    Article  ADS  Google Scholar 

  30. Chiang, I. W. et al. Purification and characterization of single-wall carbon nanotubes (SWNTs) obtained from the gas-phase decomposition of CO (HiPco process). J. Phys. Chem. B 105, 8297–8301 (2001).

    Article  Google Scholar 

  31. O'Connell, M. J. et al. Band gap fluorescence from individual single-walled carbon nanotubes. Science 297, 593–596 (2002).

    Article  ADS  Google Scholar 

  32. Bachilo, S. M. et al. Structure-assigned optical spectra of single-walled carbon nanotubes. Science 298, 2361–2366 (2002).

    Article  ADS  Google Scholar 

  33. Van Staveren, H. J., Moes, C. J. M., van Marie, J., Prahl, S. A. & van Gemert, M. J. C. Light scattering in Intralipid-10% in the wavelength range of 400–1100 nm. Appl. Opt. 30, 4507–4514 (1991).

    Article  ADS  Google Scholar 

  34. Curcio, J. A. & Petty, C. C. The near infrared absorption spectrum of liquid water. J. Opt. Soc. Am. 41, 302–304 (1951).

    Article  ADS  Google Scholar 

  35. Bashkatov, A. N., Genina, E. A., Kochubey, V. I. & Tuchin, V. V. Optical properties of human cranial bone in the spectral range from 800 to 2000 nm. Saratov Fall Meeting 2005: Optical Technologies in Biophysics and Medicine VII 6163, 616310 (2006).

    Google Scholar 

  36. Bashkatov, A. N., Genina, E. A., Kochubey, V. I. & Tuchin, V. V. Optical properties of human skin, subcutaneous and mucous tissues in the wavelength range from 400 to 2000 nm. J. Phys. D 38, 2543–2555 (2005).

    Article  ADS  Google Scholar 

  37. White, J. G., Amos, W. B. & Fordham, M. An evaluation of confocal versus conventional imaging of biological structures by fluorescence light-microscopy. J. Cell Biol. 105, 41–48 (1987).

    Article  Google Scholar 

  38. Kleinfeld, D., Mitra, P. P., Helmchen, F. & Denk, W. Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex. Proc. Natl Acad. Sci. USA 95, 15741–15746 (1998).

    Article  ADS  Google Scholar 

  39. Shih, A. Y. et al. Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain. J. Cerebr. Blood Flow Metab. 32, 1277–1309 (2012).

    Article  Google Scholar 

  40. Helmchen, F. & Kleinfeld, D. In vivo measurements of blood flow and glial cell function with two-photon laser-scanning microscopy. Methods Enzymol. 444, 231–254 (2008).

    Article  Google Scholar 

  41. Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nature Methods 2, 932–940 (2005).

    Article  Google Scholar 

  42. Dunn, A. K. Laser speckle contrast imaging of cerebral blood flow. Ann. Biomed. Eng. 40, 367–377 (2012).

    Article  Google Scholar 

  43. Boas, D. A. & Dunn, A. K. Laser speckle contrast imaging in biomedical optics. J. Biomed. Opt. 15, 011109 (2010).

    Article  ADS  Google Scholar 

  44. Vakoc, B. J. et al. Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging. Nature Med. 15, 1219–1223 (2009).

    Article  Google Scholar 

  45. Senarathna, J., Rege, A., Li, N. & Thakor, N. V. Laser speckle contrast imaging: theory, instrumentation and applications. IEEE Rev. Biomed. Eng. 6, 99–110 (2013).

    Article  Google Scholar 

  46. Hillman, E. M. C. & Moore, A. All-optical anatomical co-registration for molecular imaging of small animals using dynamic contrast. Nature Photon. 1, 526–530 (2007).

    Article  ADS  Google Scholar 

Download references

Acknowledgements

This study was supported by the National Cancer Institute of the US National Institutes of Health (NIH, grant 5R01CA135109-02, to H.D.), the American Heart Association (Innovative Science Award to C.J.K.), the NIH (grant 1R01NS064517, to C.J.K.) and a William S. Johnson Fellowship (to G.H.). The authors thank J. M. Pauly, D. Wong and T. Zhang for discussions.

Author information

Author notes

  1. Guosong Hong, Shuo Diao and Junlei Chang: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Chemistry, Stanford University, Stanford, 94305, California, USA

    Guosong Hong, Shuo Diao, Alexander L. Antaris, Changxin Chen, Bo Zhang, Su Zhao & Hongjie Dai

  2. Division of Hematology, School of Medicine, Stanford University, Stanford, 94305, California, USA

    Junlei Chang & Calvin J. Kuo

  3. Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital, Harvard Medical School, Charlestown, 02129, Massachusetts, USA

    Dmitriy N. Atochin & Paul L. Huang

  4. Department of Neurology and Neurological Sciences, Stanford University Medical Center, Stanford, 94305, California, USA

    Katrin I. Andreasson

Authors
  1. Guosong Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shuo Diao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Junlei Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alexander L. Antaris
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Changxin Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bo Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Su Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dmitriy N. Atochin
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Paul L. Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Katrin I. Andreasson
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Calvin J. Kuo
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Hongjie Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.D., C.J.K., G.H., S.D. and J.C. conceived and designed the experiments. G.H., S.D., J.C., A.L.A., C.C., B.Z., S.Z. and D.N.A. performed the experiments. G.H., S.D., J.C., A.L.A., C.C., B.Z., S.Z., D.N.A., P.L.H., K.I.A., C.J.K. and H.D. analysed the data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Calvin J. Kuo or Hongjie Dai.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 1934 kb)

Supplementary movie 1

Supplementary movie 1 (AVI 6074 kb)

Supplementary movie 2

Supplementary movie 2 (AVI 4218 kb)

Supplementary movie 3

Supplementary movie 3 (AVI 3848 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hong, G., Diao, S., Chang, J. et al. Through-skull fluorescence imaging of the brain in a new near-infrared window. Nature Photon 8, 723–730 (2014). https://doi.org/10.1038/nphoton.2014.166

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nphoton.2014.166

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing