Spatiotemporal Quantification of Endosomal Acidification on the Viral Journey

Abstract Many enveloped viruses utilize endocytic pathways and vesicle trafficking to infect host cells, where the acidification of virus‐containing endosomes triggers the virus‐endosome fusion events. Therefore, simultaneous correlation of intracellular location, local pH, and individual virus dynamics is important for gaining insight into viral infection mechanisms. Here, an imaging approach is developed for spatiotemporal quantification of endosomal acidification on the viral journey in host cells using a fluorescence resonance energy transfer based ratiometric pH sensor consisting of a photostable and high‐brightness QD, pH‐sensitive fluorescent dyes, and virus‐binding proteins. Ratiometric analysis of sensor‐based single‐virus tracking data enables to dissect a two‐step endosomal acidification process during the infection of influenza viruses and elucidates the occurrence of the fission and sorting of virus‐containing endosomes to recycling endosomes after initial acidification. This technique should serve as a robust approach for in situ quantification of endosomal acidification on the viral journey.