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

Journal name:
Nature Photonics
Year published:
Published online


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.

At a glance


  1. Imaging in various NIR subregions.
    Figure 1: Imaging in various NIR subregions.

    a, A fluorescence emission spectrum of SWNT–IRDye800 conjugate in the range of 850–1,650 nm under excitation from an 808 nm laser. The emission spectra of IRDye800 and SWNTs are plotted with different y-axis scales to accommodate both into the same graph, due to the much higher fluorescence intensity of IRDye800 than SWNTs. b, NIR fluorescence images of a capillary tube filled with SWNT–IRDye800 solution immersed at depths of 1 mm (top) and 10 mm (bottom) in 1% Intralipid, recorded in NIR-I, NIR-II and NIR-IIa regions, respectively. c, Extinction spectrum (black curve) and scattering spectrum (red curve, measured by subtracting water and Intralipid absorptions from the extinction spectrum, Supplementary Fig. 2) of 1% Intralipid in water with a path length of 1 mm measured with a UV–Vis–NIR spectrometer, as well as a reduced scattering coefficient profile (blue, converted to the unit of OD with base-10 common log) of 1% Intralipid derived from the literature33.

  2. In vivo mouse brain imaging with SWNT–IRDye800 in different NIR subregions.
    Figure 2: In vivo mouse brain imaging with SWNT–IRDye800 in different NIR subregions.

    a, A C57Bl/6 mouse head with hair removed. bd, Fluorescence images of the same mouse head in the NIR-I, NIR-II and NIR-IIa regions. In d, the inferior cerebral vein, superior sagittal sinus and transverse sinus are labelled 1, 2 and 3, respectively. e, Extinction spectra of scalp (red) and skull (blue) as well as the water absorption spectrum (black). f, Reduced scattering coefficients μ of scalp skin (red), cranial bone (blue) and brain tissue (black) plotted against wavelength, based on the previously reported scattering properties for these tissues8, 35, 36.

  3. Non-invasive, high-resolution NIR-IIa fluorescence imaging of mouse brain vasculature.
    Figure 3: Non-invasive, high-resolution NIR-IIa fluorescence imaging of mouse brain vasculature.

    a, Photograph showing the stereotactic microscopic imaging set-up. A red laser is used for alignment and shows the beam location. b, Schematic showing the penetration of NIR-IIa fluorescence through brain tissue, skull and scalp. c, Photoluminescence versus excitation spectrum of LS nanotubes in aqueous solution. The 1.3–1.4 µm NIR-IIa region is in red. d, Low-magnification cerebral vascular image taken with a field of view of 25 mm × 20 mm. e, Cerebral vascular image of the same mouse head zoomed into the left cerebral hemisphere, with a field of view of 8 mm × 6.4 mm. f, Cerebral vascular image of the same mouse head taken using a microscope objective, with a field of view of 1.7 mm × 1.4 mm. The depth of these in-focus vascular features was determined to be 2.6 mm. g, Zoomed-in image of a sub-region in f taken by a higher-magnification objective, with a field of view of 0.80 mm × 0.64 mm. Inset: cross-sectional intensity profile (black) and Gaussian fit (red) along the yellow-dashed bar. hk, Two other high-resolution cerebral vascular images with a field of view of 0.80 mm × 0.64 mm taken on another mouse (h,j) and their cross-sectional fluorescence intensity profiles (black) and Gaussian fit (red) along the yellow-dashed bars (i,k).

  4. Dynamic NIR-IIa fluorescence imaging of mouse cerebral vasculature.
    Figure 4: Dynamic NIR-IIa fluorescence imaging of mouse cerebral vasculature.

    ac, Time-course NIR-IIa images of a control healthy mouse (Mouse C1). df, PCA overlaid images showing arterial (red) and venous (blue) vessels of Mouse C1. gi, Time-course NIR-IIa images of a mouse with MCAO (Mouse M1). jl, PCA overlaid images showing arterial (red) and venous (blue) vessels of Mouse M1. m,n, Normalized NIR-IIa signal in the left (red) and right (black) cerebral hemispheres of Mouse C1 (m) and Mouse M1 (n) versus time. o, Average blood perfusion of the left cerebral hemisphere of the control group (n = 3), MCAO group (n = 4) and cerebral hypoperfusion group (n = 4), measured by the NIR-II method (red) and laser Doppler blood spectroscopy (blue). Errors bars indicate the standard deviation of each group.


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Author information

  1. These authors contributed equally to this work

    • Guosong Hong,
    • Shuo Diao &
    • Junlei Chang


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

    • Junlei Chang &
    • Calvin J. Kuo
  3. Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA

    • Dmitriy N. Atochin &
    • Paul L. Huang
  4. Department of Neurology and Neurological Sciences, Stanford University Medical Center, Stanford, California 94305, USA

    • Katrin I. Andreasson


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.

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