Real‐time Simultaneous Imaging of Acidification and Proteolysis in Single Phagosomes Using Bifunctional Janus Particle Probes

Abstract The digestion of pathogens inside phagosomes by immune cells occurs through a sequence of reactions including acidification and proteolysis, but how the reactions are orchestrated in the right order is unclear due to a lack of methods to simultaneously measure more than one reaction in phagosomes. Here we report a bifunctional Janus‐particle probe to simultaneously monitor acidification and proteolysis in single phagosomes in live cells. Each probe consists of a pH reporter and a proteolysis reporter that are spatially separated but function concurrently. Using the Janus probes, we found the acidic pH needed to initiate and maintain proteolysis, revealing the mechanism for the sequential occurrence of both reactions during pathogen digestion. We showed how bacterium‐derived lipopolysaccharides alter the acidification and proteolysis in phagosomes. This study showcases Janus‐particle probes as a generally applicable tool for monitoring multiple reactions in intracellular vesicles.