In my thesis I focus on in vivo imaging methods that can be used to study host-microbe interactions. 

We have designed a radiolabeling method that can be used to study the biodistribution of bacterial outer membrane vesicles in vivo. For our experiments, we created a novel bacterial strain, E. coli BL21.V with favorable properties for OMV production and bacterial surface display. Using this organism, we compared two different autotransporter-based surface display systems to anchor SpyCatcher on the OMV surface. Our results show that the AIDA-based system with a pET28 plasmid backbone leads to the highest number of available SpyTag binging sites on the OMV surface. Next, we developed synthetic bifunctional chelators based on SpyTag and the macrocyclic chelator NODAGA. Two SpyTag-NODAGA variants were compared (SpT-3-NODAGA and SpT-23-NODAGA). Our results indicate, that SpyTag-3-NODAGA can bind to SpyCatcher displayed on the OMV surface with higher efficiency. We have compared two 64Cu radiolabeling methods by either first carrying out the SpyCatcher-SpyTag ligation followed by NODAGA 64Cu chelation (Method 1) or first labeling SpT-NODAGA variants with 64Cu followed by SpyCatcher-SpyTag ligation (Method 2). Our results show that Method 1 can be used to prepare highly pure radiolabeled OMVs suitable for in vivo imaging. Our PET/MRI measurements show a similar OMV biodistribution in mice to previously reported data with the liver and spleen taking up most of the vesicles. Comparison with the biodistribution of labeled peptides and the results of an in vitro stability test indicate that most 64Cu stays attached to OMVs during a 12 hour investigation period. 

We also evaluated [125I]CLINME SPECT for the early detection of neuroinflammation in a murine sepsis associated encephalopathy model. We have found that following the induction of systemic inflammation by LPS injection, [125I]CLINME can be used to visualize the increase in cerebral TSPO binding sites associated with neuroinflammatory changes as early as 5 h.