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Multifunctional in vivo vascular imaging using near-infrared II fluorescence


In vivo real-time epifluorescence imaging of mouse hind limb vasculatures in the second near-infrared region (NIR-II) is performed using single-walled carbon nanotubes as fluorophores. Both high spatial (30 μm) and temporal (<200 ms per frame) resolution for small-vessel imaging are achieved at 1–3 mm deep in the hind limb owing to the beneficial NIR-II optical window that affords deep anatomical penetration and low scattering. This spatial resolution is unattainable by traditional NIR imaging (NIR-I) or microscopic computed tomography, and the temporal resolution far exceeds scanning microscopic imaging techniques. Arterial and venous vessels are unambiguously differentiated using a dynamic contrast-enhanced NIR-II imaging technique on the basis of their distinct hemodynamics. Further, the deep tissue penetration and high spatial and temporal resolution of NIR-II imaging allow for precise quantifications of blood velocity in both normal and ischemic femoral arteries, which are beyond the capabilities of ultrasonography at lower blood velocities.

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Figure 1: NIR-I and NIR-II fluorescence imaging of blood vessels in the mouse.
Figure 2: NIR-II fluorescence and micro-CT imaging of hind limb blood vessels.
Figure 3: Differentiation of femoral artery from vein in normal and ischemic mice by NIR-II imaging.
Figure 4: Differentiation of multiple arterial and venous vessels subserving a larger region of tissue.
Figure 5: Femoral artery blood velocity quantification for an ischemic hind limb and a healthy, control hind limb by NIR-II imaging and ultrasound.

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This study was supported by grants from the National Cancer Institute of the US National Institutes of Health to H.D. (5R01CA135109-02), the National Heart, Lung and Blood Institute of the US National Institutes of Health to J.P.C. (U01HL100397, RC2HL103400) and N.F.H. (K99HL098688) and a Stanford Graduate Fellowship to G.H.

Author information

Authors and Affiliations



H.D., J.P.C., N.F.H., G.H. and J.C.L. conceived of and designed the experiments. G.H., J.C.L., J.T.R., U.R., L.X. and N.F.H. performed the experiments. G.H., J.C.L., U.R., L.X., N.F.H., J.P.C. and H.D. analyzed the data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Ngan F Huang, John P Cooke or Hongjie Dai.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1,2 (PDF 1176 kb)

Supplementary Movie 1

Carbon nanotube labeled blood flow in a healthy limb. NIR-II video-rate imaging showing blood flow in a healthy hind limb of a control mouse. Total movie time: 37.5 s, frame rate: 5.3 frames/s. (AVI 5182 kb)

Supplementary Movie 2

Carbon nanotube labeled blood flow in an ischemic limb early frames. NIR-II video-rate imaging showing significantly reduced blood flow in an ischemic hind limb with occlusion in the femoral artery. The same number of frames as in Supplementary Movie 1 is shown in this movie to highlight the dramatic delay of flow into femoral artery and vein. Total movie time: 37.5 s, frame rate: 5.3 frames/s. (AVI 6168 kb)

Supplementary Movie 3

Carbon nanotube labeled blood flow in an ischemic limb late frames. NIR-II video-rate imaging showing significantly reduced blood flow in an ischemic hind limb with occlusion in the femoral artery up to 247.5 s post injection of carbon nanotubes. Total movie time: 247.5 s, frame rate: 0.89 frames/s. (AVI 7240 kb)

Supplementary Movie 4

Differentiation of arterial and venous vessels subserving a larger area. NIR-II video-rate imaging showing different blood flow behaviors of arterial and venous vessels subserving a larger area than just the hind limb, which is the basis for differentiation of blood vessel type based on principal component analysis. Total movie time: 31.9 s, frame rate: 5.3 frames/s. (AVI 3718 kb)

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

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