Discrimination of nanovesicles using two-color laser-induced fluorescence detection

A two-color fluorescence-detection system was developed to distinguish between two types of nanovesicles labeled with different fluorescent dyes. This system employs two lasers emitting at distinct wavelengths (488 and 635 nm). These lasers are reshaped into sheet-like beams and focused at separate positions within a square capillary. As vesicles flow through the capillary, they pass through the laser beams and emit fluorescence when excited by the corresponding wavelength. This technique allows for the counting of vesicles and identification of the specific fluorophores on the vesicles based on their emission positions. The system was validated using liposomes labeled with fluorophores excited by the 488 and 635 nm lasers. It was then applied to differentiate CD63-positive extracellular vesicles (EVs) from other types of EVs in a culture media consisting of HeLa and A549 cells. Additionally, the system was used to study the effect of the anticancer drug doxorubicin on EV secretion in these cancer cells.