Near-Infrared Photoluminescent Carbon Nanotubes for Imaging of Brown Fat

Figures

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  Figure 1. Schematics of CNT, PMB, and the supposed structure of PMB-coated CNT (a). PL spectra of PMB-CNT dispersed in water (b). The black rectangle in (b) denotes the excitation and PL wavelength region used in the imaging. Typical NIR-PL images of a mouse 5 h after i.v. injection of PMB-CNT (c–e) and those of a control mouse without injection (f–h). Is, interscapular; Pv, paravertebral; Ax, axillar; Rp, retroperitoneal; In, inguinal; Li, liver; Pe, pelvic; Bo, bowels. The PL intensity ranges are indicated in the bottom left of each image.

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  Figure 2. Histology of bright PL-emitting tissues. Adipose tissues from interscapular (a), axillar (b), inguinal (c), and paravertebral (d) areas of PMB-CNT-injected mice at a post-injection time of 3.5 h. Staining: HE (left), UCP-1 (centre), and NFR (right). Visible-light micrographs (left and centre) and NIR-PL micrographs (right).

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  Figure 3. Detailed examinations of adipose tissues with CNT-accumulations. NIR-PL images (A) and naked-eye observations (B) of a paraffin block with a tissue cross-section from iBAT to skin. The tissue was collected from a PMB-CNT injected mouse at 3.5 h post-injection. Multi-field low magnification micrographs of the tissue slices stained with nuclear fast red (C) or an anti-UCP-1 antibody (D) are shown. High magnification NIR-PL micrographs (E, 1e-5e) and visible-light micrographs for UCP-1 staining (F, 1f-5f) are also presented.

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  Figure 4. (A) Visible-light micrographs (a,e,i), NIR-PL micrographs (b,f,j), and merged images (c,g,k) with higher magnification (d,h,l), near an arteriole (a–d) and a venule (e–h) in iBAT, and near an arteriole/venule in iWAT (i–l). The tissues were obtained from mice at 3.5 h post-PMB-CNT injection (n = 3). Bright objects in NIR-PL images correspond to CNTs. Tissues were stained with NFR. (B) A confocal microscopy image, NIR-PL micrograph, and merged image of iBAT, obtained from a male mouse at 3 h post-PMB-CNT injection (n = 1). Endothelial cells were visualised by immunostaining with anti-CD31 antibody. (C,D) TEM observations to determine the localisation of CNTs in iBAT (C) and iWAT (D), obtained from a mouse at 3.5 h post-PMB-CNT injection (n = 1). Red arrows in (C) and an area enclosed by a red dotted line in (D) denote the existence of CNTs.

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  Figure 5. The specific interaction between PMB-CNTs and apolipoproteins. (A,B) Western blotting using the reaction of serum/PMB-CNTs (lane 1) and an equivalent volume of serum (lane 2) to detect albumin (a), ApoB (b), and ApoE (c). Albumin Western blotting was performed using 1/10 diluted samples. (B) Fluorescence images of PMB-CNT/serum mixture-smeared glass slides immunostained using control IgG (a, c: normal rabbit, e: normal goat), anti-ApoB antibody (b), anti-ApoE antibody (c), and anti-ApoC antibody (f). (C) TEM images of the liver of a PMB-CNT-administered mouse. Fine CNT structures were detected within lipid droplets (LDs) of stellate cells: LD(1) and LD(2) in (a–c), and LD(1), LD(2), and LD(3) in (d–f). CNTs were not observed in LD(4) and LD(5) in (d). HSC: hepatic stellate cell. Red and blue broken-lines denote HSC and the area where CNTs present, respectively. (D) A model for the binding of the CNT-PMB-apolipoprotein complex, with its receptor on the capillary endothelial cells of adipose tissue.

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  Figure 6. NIR CNT-PL dynamics in vivo. (A) Time course of whole body NIR PL images of a PMB-CNT-administered mouse (a–h), PL intensities in iBAT (red), a non-BAT area in the scapula (blue), and background (black). (i) PL intensities were measured at two to four places in each area. Error bars denote standard errors. Similar results were obtained from other mice (n = 5). (B) NIR PL micrographs of iBAT (a, b) and iWAT (Beige; c, d) at 3.5 hours (upper panels) and 14 days (bottom panels) post-injection (n = 3). (C) TEM images of iBAT (a) and iWAT (b) 14 days after injection (n = 1).