Localizing Extracellular Vesicles by TREx Microscopy In Vitro and In Vivo

Abstract

In this report we present our data for the use of TREx microscopy, an improved expansion microscopy protocol, to visualize extracellular vesicles (EVs) in vitro and in vivo. Here we focused specifically on exosomes. In vitro visualization was done by combining two PM bound MVB markers, ORP1L and Arl8b, with exosome reporter CD63-pHluorin. Here, we found that ORP1L was a better candidate than Arl8b, which caused MVB anomalies. TREx was used to visualize precursor-exosomes, otherwise known as ILVs, with CD63-pHluorin contained in PM bound ORP1L MVBs. Visualization of EVs in vivo was was achieved by adaptation and optimization of the TREx protocol for a zebrafish animal model. The main challenges revolved around the optimization of the gel composition to properly embed the fish in the gel and the optimization of expansion kinetics due to tissue heterogeneity which can cause distortions in the sample. Imaging efforts were focused on the caudal vein plexus area of the zebrafish were EVs accumulate. The optimized TREx protocol allowed the visualization of single EVs inside the zebrafish which provided more insight on EV fates in vivo with higher resolution than previously acquired. Overall, our research highlights the great potential of TREx microscopy in the field of EVs.