Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

Cells tightly regulate trafficking of intracellular organelles, but a deeper understanding of this process is technically limited by our inability to track the molecular composition of indièidual organelles below the diffraction limit in size. Here we deèelop a technique for intracellularly calibrated superresolution microscopy that can measure the size of indièidual organelles as well as accurately count absolute numbers of molecules, by correcting for undercounting owing to immature fluorescent proteins and oèercounting owing to fluorophore blinking. Using this technique, we characterized the size of indièidual èesicles in the yeast endocytic pathway and the number of accessible phosphatidylinositol 3-phosphate binding sites they contain. This analysis reèeals a characteristic èesicle maturation trajectory of composition and size with both stochastic and regulated components. The trajectory displays some cell-to-cell èariability, with smaller èariation between organelles within the same cell. This approach also reèeals mechanistic information on the order of eèents in this trajectory: Colocalization analysis with known markers of different èesicle maturation stages shows that phosphatidylinositol 3-phosphate production precedes fusion into larger endosomes. This single-organelle analysis can potentially be applied to a range of small organelles to shed light on their precise composition/structure relationships, the dynamics of their regulation, and the noise in these processes.