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

Most cell types release a diverse array of extracellular vesicles (EVs) that contribute to intercellular communication. In particular, considering the heterogeneity of EVs, methods capable of identifying and measuring individual vesicles are limited. Here, we used fluorescence and colocalization Nanoparticle Tracking Analysis (NTA) to identify the subcellular origin of vesicles and determine their physical characteristics, as well as colocalization ratios of endoplasmic (ER) and Mitochondria (Mito) positive EVs in human choriocarcinoma cells (JAr) and bovine follicular fluids (BFF). The labeling efficiency for ER-labeled JAr EVs purified in SEC was 67.11 ± 25.40%, compared to 96.27 ± 13.72% of BFF EVs. Regarding Mito dye labeling efficiency, SEC-purified BFF EVs (14.21 ± 7.45%) provided lower Mito-positive fluorescent particles than JAr EVs (25.74 ± 4.46%). The proportion of CellMask Deep-Red (CMDR) membrane labeling of nanoparticles varied across JAr and BFF EVs. Furthermore, colocalization analysis of ER and Mito-dye-labeled JAr and BFF revealed potential intracellular interactions between organelles and the EV biogenesis pathways. The integration of novel colocalization technology into fluorescence-NTA (F-NTA) represents a significant advancement in the field of single EV particle analysis in deepening our understanding of EV biology.