The aim of the present study was to explore the physicochemical parameters that influence the performance of Tzs for pretargeted brain imaging and apply this knowledge to develop a suitable imaging agent for this purpose (Figure ). We decided to develop 18 F-radiolabeled Tzs. First of all, fluorine-18 ( 18 F) is widely considered to be the ideal PET radionuclide due to its convenient half-life (110 minutes), high positron branching ratio (97%) and short positron range in tissue (max. range 2.4 mm in H2O). These properties altogether result in high-resolution images with low patient radiation dose. Secondly, F can be centralized produced in large scale and 18 F-labeled radiopharmaceuticals consequently distributed for clinical use. Thirdly, introduction of 18 F into the wider Tz-framework can be used to fine-tune its reactivity while simultaneously, the physiochemical properties of the fluorinated framework can be manipulated with a second handle (Figure ). We have recently developed methods to prepare highly reactive Tzs labeled with fluorine-18. These methods were used to label all Tzs in this study. The respective development strategy is displayed in Figure . In short, we designed and synthesized a panel of 18 F-labeled Tzs guided by parameters reported to increase the chances to successfully develop a PET brain tracer. Selection of the parameters was inspired by the BBB score -a parameter developed to identify brain-penetrating molecules. Subsequently, the Tz panel was subjected to in vitro and in vivo screening. Based on the results, a subset of Tzs was selected for a final evaluation round in a pretargeted model. In this model, rats were intracerebrally injected with a noninternalizing TCO-polymer. This polymer was then -in a second step -targeted by selected Tzs. We decided to use this invasive model as it allows to solely study the in vivo performance of the Tz without challenges arriving from a brain targeting antibody, for example with respect to blood circulation, target engagement or metabolism. Obtained results were afterwards analyzed to identify possible relations between the in vivo performance and physicochemical parameters of the Tzs. The aim of this work was to identify a Tz most suited for pretargeted brain imaging.