EV imaging series: Using super-resolution to characterize single-EVs

Flow-based methods have traditionally been used to size and measure the concentrations of EV populations and although more sophisticated methods are now available for studying EVs on a single-vesicle level, traditionally these methods had relatively low accuracy and struggled to discriminate between EV subpopulations. When it comes to analysing single EVs, a recent Twitter poll showed that electron microscopy is still the most popular choice of method. With its ability to image EVs at very high resolution, EM has some clear advantages when it comes to single EV characterization but falls short in its capability to visualize multiple markers simultaneously. In contrast, conventional fluorescence imaging techniques offer a simple means of characterizing EV sub-populations based on the presence or absence of multiple biomarkers within a single experiment but suffer from the diffraction-limit of light, restricting their usefulness in accurately sizing EVs or visualizing biomarker distributions.

So is there another alternative? – One that compares with the accuracy of EM for precise sizing but allows for the detection of multiple biomarkers with minimal sample preparation? Throughout this series, we hope to convince you of the benefits of super-resolution microscopy for EV characterization, a very powerful but underused technique in the field. Using single-molecule localization microscopy (SMLM), images of EVs can be acquired at a resolution of 20 nm to size and characterize the spatial distributions of biomarkers at the nano-scale.