Serial =me-lapse images captured the brightly AZ647-labeled RBCs at baseline and highly efficient iTCO cleavage dynamics, while control staining in the PE channel remained stable throughout (Fig. , Supplementary Fig. , and Supplementary Movie S1. RBC brightness began to decline rapidly within 15-20 seconds of Tz administra=on and was indis=nguishable from background three minutes later. AZ647 signal intensity vs. =me is well-fibed by a single exponen=al decay with a first order rate constant of 0.021 s -1 , corresponding to a cleavage half-life of just 33 seconds. The observed click-to-release in the murine vasculature thus proceeded markedly faster than the cuvebe-based reac=on in plasma (cf. Fig. ). Although some component of this accelera=on may arise from inherent differences between cleavage of the AZ647-iTCO linker and AMC release, the varying reac=vity as a func=on of hydrophobicity for various Tz and TCO tools suggests that the hydrophilic nature of the intravital microscopy probes is relevant to the excep=onal performance that we observe for iTCO in vivo.