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Supplementary figures in the manuscript.

Anti-vascular endothelial growth factor (anti-VEGF) therapy is effective for reducing the severity level of diabetic retinopathy (DR). However, it is difficult to determine the in vivo spatial and temporal expression of VEGF in the DR retina at an early stage. Here, we report a quantitatively fluorescence molecular imaging and image analysis method by creating a VEGF targeted fluorescence imaging probe, which can potentially detect and predict anti-VEGF treatment response. Moreover, the ex vivo multiscale fluorescence imaging demonstrated the spatial correlation between VEGF relative expression and vascular abnormalities in two and three dimensions. It revealed that VEGF was mainly abnormally expressed at the bifurcation of the microvessels, which advances the knowledge of the DR progression by molecular fluorescence imaging. Our study has the potential to achieve early detection of DR disease, provide more insight into understanding anti-VEGF treatment, and may help stratify patients based on the molecular imaging of retinal VEGF.