To interrogate the in vivo performance of iTCO, we designed a method that uses confocal microscopy to monitor real-=me probe cleavage from the surface of circula=ng cells. Our strategy leverages intravital imaging of the murine ear vasculature, which is capable of genera=ng high resolu=on images on the singlecell level while avoiding the invasiveness of dorsal window chambers or other surgical imaging procedures (Fig. ). To minimize confounding biological effects from ac=ve cellular internaliza=on and/or other immunologic uptake mechanisms, we selected red blood cells (RBCs) as a model system and the panerythrocyte lineage marker TER119 for an=body detec=on . The sheer abundance of RBCs facilitates quan=ta=ve analysis, as the cells of interest-in contrast to leukocyte subsets-are both highly uniform and con=nuously in view, allowing robust popula=on averaging over the experimental =mescale.