To evaluate the in vivo click performance of our Tzs, we used an invasive model based on intracerebral injection of a TCO-functionalized polymer (Figure ). As mentioned beforehand, this model circumvents challenges that arise when administering a targeting vector systemically. For example, no blood circulating mAb is present in the intracerebral injection model that could 'click away' a significant fraction of administered Tz. A detailed description of the model is reported in the Supplementary Information. In brief, Long-Evans rats were injected with TCO-decorated PeptoBrush polymer (100 µg polymer, 72 nmol TCO in 4 µL of 0.1M phosphate buffer at pH 7.2) into the right striatum. This polymer has been shown to be noninternalizing. Moreover, in rodent plasma, which is similar to interstitial fluid of the brain, at least 50% of the TCOs were stable for 24 h. Restoration of the BBB integrity 20 h after polymer injection was confirmed by MRI imaging with the gadolinium-based contrast agent ProHance (SI). To determine the retention of the injected TCO-PeptoBrush at the injection site, we labeled the polymer with indium-111 (100-200 kBq/mg polymer, SI Section S10). 20-24 h after TCO-polymer injection, rats were injected with 18 F-Tzs (10-30 MBq, 0.3-1.3 nmol) into the lateral tail vein and scanned in a PET scanner for 90 min. Radioactivity uptake in the right striatum was compared with the uptake in the left (polymer-free) striatum. After the scan, rats were sacrificed, their brains dissected into right and left hemispheres and gamma-counted 24 h later (to measure the 111 In-counts after the decay of 18 F). On average, 49.4±8.7% (n=14) of the injected 111 In-activity was found in the injected brain hemisphere, while only 0.5±0.2% (n=14) was found in the control hemisphere, indicating good and consistent retention of the TCO-polymer (SI Figure ). It is not known whether the observed loss of 111 In-activity from the injection site reflects de-chelation of 111 In or washout of the whole TCO-polymer. However, activity loss from the injection site was spread throughout the whole body. Therefore, local TCO concentration in the striatum is >1000-fold higher than elsewhere in the body (SI). Preferential 18 F-uptake in the TCO-polymer pre-injected striatum was clearly visible for all investigated Tzsboth on summed images as well as on detected TACs (Figure ). We ranked 18 F-Tzs in terms of their absolute and relative imaging contrast (Figure ). Relative imaging contrast was defined as the ratio between 18 Fuptake in the TCO-polymer injected vs the non-injected striatum, while the absolute contrast was defined as the difference between the uptake values, expressed in %ID/mL. The best relative contrast was observed for [ 18 F]10, the best absolute contrast for [ 18 F]1 -however, with a huge variation from scan to scan. [ 18 F]18 showed high values in both categories and is because of this our prime candidate to be used in further studies. These results are not expected from our prediction model displayed in Figure . For example, [ 18 F]1 was suggested to be the least promising compound whereas good in vivo click uptake was observed in pretargeted experiments.