Representative NIRF imaging of different groups 24 h after infusion of the fluorescent probe.

<p>In vivo NIRF imaging (A, Left) or ex vivo NIRF imaging of the mouse brains removed from the skull (A, Right) showed no difference between the hemispheres in sham-operated mice injected with the A15 probe. The rectangular boxes represented the ROIs placed over the right and left hemisphere. There was only slight increase of fluorescence intensities in the injected hemisphere compared with the contralateral side in MCAO mice injected with the C15 control probe (in vivo NIRF imaging, B, Left; and ex vivo NIRF imaging, B, Right). Strong fluorescence was seen over the ipsilateral side of MCAO mice injected with the A15 probe shown by in vivo NIRF imaging (C, Left) or ex vivo NIRF imaging (C, Right). The images were normalized on the color scaling bar. Target-to-background ratios (TBRs) were calculated from ROI analyses of noninvasive NIRF images in different groups as shown in Fig D. Only the MCAO mice receiving the A15 probe showed significantly higher TBRs (*P<0.05 versus SHAM). Bar in (A–C) = 5 mm.</p

Fluorescence images of brain sections showed the ipsilateral cortex of MCAO mice at 1, 8, 24 and 96 hours after intravenous injection of A15.

<p>The green signals showed the staining with FITC-labeled antibodies against fibrin (A), and the red signals (NIRF channel) displayed the distribution of the injected Cy5.5-labeled probes (B). The embedded scatter gram in the upper left corner of each image (C) showed good overlap of colocalization analysis (C). (40×, Bar = 50 µm).</p

Distribution of the injected A15 probe 24 h after cerebral ischemic induction.

<p>Areas with high fluorescence intensities were observed over the ischemic region in MCAO mice that received A15 (A), corresponding to the pallor in TTC staining (B). High magnification of the boxed region in A was shown in C (C, 40×). Intense and diffuse Cy5.5 fluorescence from A15 was observed in the ischemic area of the cortex and nearby micro-vessels (↑), whereas fluorescence was scarcely visible in the nonischemic areas. The border between the ischemic and nonischemic area was clearly delineated after TTC (B) and hematoxylin and eosin staining (D).</p

Time courses of in vivo NIRF imaging of MCAO mice injected with A15 at 6 h, 8 h, 20 h and 96 h (A).

<p>Quantification of target-to-background ratios (TBRs) at different time points (From 0–96 h post injection of A15) was shown in B. The group that were significantly different were compared with 0 h (TBRs = 1). (*P<0.05 versus time 0).</p

Quantitative analysis of targeted signals from in vivo and ex vivo NIRF imaging of the mouse brains removed from the skull and lesion volume of T₂WI (%HLV^e).

<p>The targeted signals of in vivo and ex vivo NIRF images are separated from background signals by multispectral imaging technology (A). ROI was selected by threshold segmentation (B). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030262#pone-0030262-g005" target="_blank">Fig. 5C</a> shows T₂WI of MCAO mice 24 h after injection of A15, and area of hyperintensity delineates ischemia lesion. A significant correlation is observed between the total targeted signal values of in vivo NIRF images (R² = 0.526, P<0.05) and the total targeted signal values of ex vivo NIRF images of MCAO mice injected with the A15 probe (D, Left). A significant correlation is observed between the value of total targeted signal from in vivo NIRF imaging (R² = 0.483, P<0.05) or from ex vivo NIRF imaging (R² = 0.843, P<0.05) and lesion volume of T2WI (%HLV^e) (D, Middle, Right). Bar in (A–D) = 5 mm.</p