Introduction Thrombus formation occurring in atherosclerotic plaques are responsible for plaque complication and acute events such as myocardial infarction. Pro-thrombotic factors expressed within the lesions therefore constitute relevant biomarkers for the imaging of prone-to-thrombus lesions. Tissue factor (TF), the most potent trigger of the coagulation cascade, plays a major role in atherothrombosis. Our objective was to generate and characterized single-domain antibody (sdAb) based radiotracers targeting murine TF (mF) for the noninvasive imaging of atherosclerosis in mice. Methods Seven sdAbs were produced and SPR was performed to assess their affinity. Then, following radiolabeling with 99mTc, rapid in vivo screening was performed using mice bearing mTF positive B16F10 tumors (n=36). The 3 most potent candidates allowing the in vivo SPECT imaging of mTF were further evaluated in ApoE-/- mice fed a high fat diet for 20 weeks (n=21). An irrelevant sbAb (R3b23) and a previously validated sdAb directed against the inflammatory marker VCAM-1 (cAbVCAM1-5) were employed as negative and positive controls, respectively. In vivo competition study was performed to validate the specificity. TF expression was determined in various organs by ELISA. Autoradiography (ARG) was performed on 20µm frozen slices of the aorta to investigate the tissular distribution. Results/Discussion All 7 sdAbs showed excellent affinities for their target, with a KD ranging from 0.02 to 82.4 nM. TF-positive tumors were successfully visualized by SPECT/CT imaging. More specifically, the sdAb 99mTc-66A12 demonstrated the highest uptake in tumor (3.3±0.2%ID/g) and target-to-blood ratio (10.7±2.8). Specific uptakes were also observed in most investigated organs. The biodistribution of the 3 leads compound on ApoE-/- mice was in accordance with the expression profile of TF. ARG quantification revealed that, in the aortic sinus where atherosclerosis develops, no significant difference in uptake was observed between the anti mTF and the irrelevant sdab (1.1±0.1%ID/g vs 0.5±0.1%ID/g for 99mTc-66A12 and 99mTc-R3B23, respectively), while 99mTc-cAbVCAM1-5 uptake was significantly higher (2.5±0.3 %ID/g, P>0.01). Surprisingly, higher uptake was observed in the abdominal aorta that is devoid of atherosclerotic lesions. Consequently, atherosclerotic lesions were not detectable in vivo by SPECT. Conclusions sdAb-based radiotracers targeting tissue factor were successfully developed. In vivo, they specifically bound to mTF-positive tumors and mTF-positive organs, that were therefore readily visible by SPECT imaging. However, in the ApoE-/- mice model, the suboptimal uptake in lesions in comparison to normal aortic wall did not allow atherosclerosis imaging by SPECT.