Phenotypic analysis of extracellular vesicles: a review on the applications of fluorescence

Abstract

Extracellular vesicles (EVs) have numerous potential applications in the field of healthcare and diagnostics, and research into their biological functions is rapidly increasing. Mainly because of their small size and heterogeneity, there are significant challenges associated with their analysis and despite overt evidence of the potential of EVs in clinical diagnostic practice, guidelines for analytical procedures have not yet been properly established. Here, we present an overview of the main methods for studying the properties of EVs based on the principles of fluorescence. Setting aside the isolation, purification and physicochemical characterization strategies which answer questions about the size, surface charge and stability of EVs (reviewed elsewhere), we focus on available optical tools that enable the direct analysis of phenotype and mechanisms of interaction with tissues. In brief, the topics on which we elaborate range from the most popular approaches such as nanoparticle tracking analysis and flow cytometry, to less commonly used techniques such as fluorescence depolarization and microarrays as well as emerging areas such as fast fluorescence lifetime imaging microscopy (FLIM). We highlight that understanding the strengths and limitations of each method is essential for choosing the most appropriate combination of analytical tools. Finally, future directions of this rapidly developing area of medical diagnostics are discussed.

Keywords: Exosomes; FLIM; FRET; cancer; fluorescence depolarization; fluorescence lifetime; fluorescent dye; fluorescent protein; microarrays; microfluidics; microscopy; nanobody; nanoparticle tracking analysis; qPCR; quantum dot; super resolution microscopy.

Figure 2.

Schematic representation of the relative sizes of EVs and types of fluorescent labels and molecular probes.

Figure 1.

Simplified schematic overview of the main optical techniques used for EV analysis. Centre image: EVs secreted from cells. Techniques where EVs are analysed in suspension include A: nanoparticle tracking analysis, B: flow cytometry, C: fluorescence spectroscopy (lifetime, polarization). Methods where EVs are immobilized on a surface include D: fluorescence microscopy for isolated EVs (for live imaging, EVs can be in suspension) and E: microarrays and microfluidics. Also, F: quantitative PCR, in which the EVs are lysed.