Analysis of Extracellular Vesicle Data on Fluorescence and Atomic Force Microscopy Images.

Extracellular vesicles (EV) enable cell-to-cell communication in the body of an organism and carry significant potential in the medical field as disease indicators, tissue regeneration, and drug carriers. We developed two workflows to analyze EV data based on microscopy images reliably. The first workflow enables determining the total number of fluorophores per EV by analyzing the photobleaching step counts in a stepwise photobleaching experiment recorded through fluorescence microscopy. Furthermore, we present a workflow for the quality assessment of EV populations through multimodal imaging. Thus, enabling quantification of the purification quality (differentiation between EVs and other components) and the labeling ratio. Both workflows have shown excellent results on various data sets and under various conditions.