The NIR-I absorption and NIR-II emission made TPE-BBT a potential candidate for NIR-II bioimaging in vivo. Here we evaluate the performance of TPE-BBT PLNPs for NIR-II vascular imaging. The DLS spectrum and TEM image proved that TPE-BBT PLNPs had a homogeneous spherical structure (d < 30 nm) (Figure ). Besides, the cytotoxicity assay using human umbilical vein endothelial cells (HUVEC) demonstrated that TPE-BBT had negligible toxicity even when the molecular concentration reached 1 mg/ml (Figure ). In addition, both TPE-BBT and TPEO-BBT PLNPs showed superior photostability to the commercial NIR contrast agent indocyanine green (ICG) (Figure ). These results confirmed the potential of TPE-BBT in biological applications. TPA-BBT PLNPs were also prepared, and the NIR-II imaging qualities in mouse models were compared using TPE-BBT PLNPs, TPA-BBT PLNPs, and ICG, respectively. The PLNPs and ICG were intravenously injected (100 μL, molecular concentration = 1 mg/mL), and an InGaAs camera recorded the angiographies with 850 and 1000 nm long-pass (LP) filters (Figure ). All three dyes showed good imaging clarity of blood vessels in the hindlimbs, while TPA-BBT PLNPs showed better imaging resolution than ICG, which was consistent with our previous conclusions. TPE-BBT PLNPs demonstrated the best imaging quality, with the smallest full width at half maximums (FWHM) of 0.23 mm and the highest SBR of 1.76 (Figure ). This result implied that higher brightness in the NIR-II window is essential for better imaging quality. Molecular concentration: 1 mg/mL; λ ex = 808 nm; power density = 30 mW cm 2 ; 850 and 1000 nm LP filter; exposure time = 100 ms. (gi) Cross-sectional fluorescence intensity profiles along the red lines in blood vessels treated by (g) TPE-BBT PLNPs, (h) TPA-BBT PLNPs, and (i) ICG, respectively. Gaussian fits are shown in red dashed lines.