We used a model of focal atherosclerotic-like plaques in the femoral arteries (located to 1.5 cm from skin surface) of New Zealand white rabbits. All animal studies performed were approved by the Washington University Animal Studies Committee. Endothelial denudation of surgically exposed right femoral artery was induced by air desiccation of the luminal surface as described previously.4 The uninjured left femoral artery served as the internal control. The animals were maintained on a cholesterol-enriched diet ( in blood), and over time the air desiccation led to a focal lesion. In this animal model, anatomical coregistration was not used because the location of the lesion was apparent due to the surgical markings and identification by the surgeon at each imaging session. In a clinical setting, coregistration of the FMT probe with the ultra sound (US) probe can be utilized. We chose to monitor the progression of the receptor, NPR-C, which has been shown to undergo changes during atherosclerotic plaque progression and was recently evaluated as a PET imaging marker.5 NPR-C is a cell surface protein found on endothelial, vascular smooth muscle, and macrophage cells. Natriuretic peptides (NPs) play an important role in regulating cardiovascular homeostasis. NPR-C (clearance receptor) removes NPs from circulation by receptor mediated endocytosis.6 We evaluated a bioconjugate LS668 (Cypate-RSSc[CFGGRIDRIGAC]), consisting of a near infrared (NIR) fluorescent dye cypate conjugated to a targeting peptide, C-type atrial natriuretic peptide, specific for NPR-C [Fig. 1(a)]. NIR fluorescent (700 to 900 nm) imaging agents are desirable for in vivo imaging due to enhanced depth penetration of NIR light and low tissue autofluorescence.7 Cell studies demonstrated that LS668 (, 30-min incubation at 37°C, 5% ) was selectively internalized by stably transfected 293T-NPR-C cells [Fig. 1(b)].8 Internalization was blocked in the presence of excess () C-ANF peptide [Fig. 1(c)], and additionally, LS668 did not internalize into the control 293T-NPR-A cells [Fig. 1(d)], supporting receptor mediated endocytosis of LS668. For in vivo imaging, 24-h postinjection of LS668 was selected as the optimal time point as clearance of LS668 from blood was achieved at 24 h. Both injured and control femoral arteries of three animals were imaged at day 3 and weeks 1, 2, 4, 6, and 8 following the surgery. For each time point, LS668 ( intravenous) was injected and 24 h later FMT scans were performed (5 min each) in triplicates for each artery. FMT reconstruction was performed as reported earlier to obtain three-dimensional (3-D) data from the tissue containing the lesion.3 FMT scans of the respective arteries before surgery were used as blank scans for image reconstruction. In the reconstructed data, localized fluorescence signal indicating accumulation of LS668 was observed at a depth of 4 to 16 mm and over 15 mm of length consistent with the location of the focal lesion. Coronal section images of the 3-D volumes are shown at a depth of 7 mm for one representative animal [Fig. 2(a)]. The corresponding sagittal and transverse sections show the spread of the lesion along the length and breadth [Figs. 2(a) and 2(b)]. Near background signal from the tissue surrounding the localized fluorescent region indicated negligible nonspecific uptake by surrounding tissue. Contralateral noninjured femoral arteries showed minimal signal indicating negligible background uptake of LS668 in the control artery. Integrated fluorescence signal (directly related to the quantity of LS668 in tissue) was calculated from each tissue volume (thresholded at above 20% of respective maximum signal) [Fig. 2(c)]. Unpaired test (two tailed) showed statistically significant difference between control and injured arteries at week 2 () and week 6 () [Fig. 2(c)]. The changes in the signal over time most likely result from a transient increase in macrophages after injury usually at week 2, followed by a decrease resulting from the increased amount of the matrix and decrease in cellularity. Inflammation resumes in the following days or weeks due to the diet-induced macrophage-enriched unstable lesions. The variance within the cohorts highlights the differences in the pace of lesion formation in individual animals, probably as a function of their cholesterol levels. Ex vivo tissue biodistribution even after 24-h postinjection at week 8 showed -fold () higher localization of LS668 into the injured femoral artery as compared to the control femoral artery [Fig. 2(d)].