# Detection of Organelle-Specific Dyes Labeled Extracellular Vesicles with Colocalization-Fluorescence Nanoparticle Tracking Analysis

## TL;DR

This paper introduces a new method using fluorescence and colocalization nanoparticle tracking analysis to study the origin and characteristics of extracellular vesicles from different sources.

## Contribution

The integration of colocalization technology into fluorescence-NTA enables detailed single EV particle analysis.

## Key findings

- ER-labeled BFF EVs showed higher labeling efficiency than JAr EVs.
- Mito-positive labeling was lower in BFF EVs compared to JAr EVs.
- Colocalization analysis suggests interactions between organelles and EV biogenesis.

## Abstract

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.