EV imaging series: Using super-resolution to characterize single-EVs
Welcome to our latest Extracellular Vesicle (EV) imaging series!
This is the first of three scheduled posts that will be coming to you over the next few weeks. Here, we will discuss the latest characterization trends and open questions researchers are facing within the EV field. We have created this imaging series in the hope that we can share the awesome research that is currently on-going, along with the incredible technology that is fueling it.
The field of EV research has exploded over the past few decades with a rapidly growing list of compelling publications behind it. However, until MISEV published their guidelines for EV characterization in 2014, the field suffered from a real issue with data reproducibility owing to the wide variety of isolation, purification and characterization techniques that were being used. To date, researchers commonly use flow-based characterization methods, fluorescence imaging techniques and electron microscopy (EM) and whilst they all come with their own set of advantages, they are not without their limitations.
Feel free to share our imaging series on social media, and if you would like to learn more about EVs and single-molecule localization microscopy, please visit our EV hub using the link below.
Learn more about EV characterization using single-molecule localization microscopy by reading our application note.
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.
3-color widefield vs dSTORM image of CD9-ATTO488, CD63-Alexa Fluor®-647 and CD81-Alexa Fluor®-555 labeled EVs from isolated HCT116 cell line on the Nanoimager. Move the slider to see the difference.
To learn more about how we are applying the SMLM techniques of the Nanoimager to characterizing heterogeneous populations of EVs, make sure you download our application note using the link below. Also be sure to check out our EV hub that has a wealth of other useful imaging guides and microscopy tips. ONI is dedicated to providing researchers with access to the most advanced and precise tools available, to accelerate science and fight disease.
Thanks for reading, and we look forward to seeing you back here for our next post. If you have any questions or suggestions, or would simply like to get in touch to say hi, please don’t hesitate to contact us (email@example.com). Please feel free to share this imaging series post on your social media and help spread the knowledge!