Detection of organelle-specific dyes labeled extracellular vesicles with colocalization-fluorescence nanoparticle tracking analysis

Cells constantly release extracellular vesicles, including exosomes, to communicate with their surroundings. These tiny particles carry proteins, lipids, and nucleic acids that reflect where they come from inside the cell. While extracellular vesicles are known to be highly diverse, most analytical methods still measure them in bulk, making it hard to understand how individual vesicles differ from one another.

Researchers at the University of Tartu, Estonia describe a way to overcome this limitation by combining fluorescence labeling with nanoparticle tracking analysis. Their approach focuses on identifying where individual vesicles originate inside the cell and measuring their physical properties at the single-particle level.

The team used fluorescent dyes that specifically label the endoplasmic reticulum and mitochondria, two key cellular organelles. By tagging vesicles with these dyes and analyzing them using fluorescence nanoparticle tracking analysis with colocalization, the researchers could determine whether individual extracellular vesicles carried markers from one or both organelles. This allowed them to link vesicles back to their subcellular origins.

Experiments were performed using vesicles released from human choriocarcinoma cells and from bovine follicular fluid. The results showed clear differences in labeling efficiency and organelle-associated vesicle populations between the two systems. Some vesicles carried markers linked to the endoplasmic reticulum, others to mitochondria, and a subset showed evidence of overlap, suggesting coordinated interactions between organelles during vesicle formation.

Importantly, the study demonstrates how adding colocalization analysis to fluorescence nanoparticle tracking analysis improves the resolution of extracellular vesicle measurements. Instead of treating vesicles as a uniform population, researchers can now examine how individual vesicles differ in origin and composition.

This work provides a practical tool for studying extracellular vesicle heterogeneity and sheds light on how vesicle biogenesis may be linked to intracellular organelle interactions. Such insights are valuable for basic cell biology and for applications where vesicles are explored as biomarkers or therapeutic carriers.