Fluorescent Labeling of Extracellular Vesicles
Characterizing EV s is of paramount interest for people thinking in diagnostic applications. Measuring the size of nanoparticles is, of course, one of the features that people look into, the other are the specific phenotypes of the vesicles. For this, fluorescence is an ideal tool, because it can target specific proteins within a distribution of particles. The key objective is to study phenotype or epitope prevalence.
A by-product of fluorescent labeling is that nanoparticle tracking analysis can also benefit from it. Therefore a robust protocol can be beneficial not only for phenotyping but also for improve performance of the sizing method. A paper published on Scientific Reports 1 developed a protocol to study the effect that labeling with quantum dots has on the final result. They also looked into how some purification strategies alter the results.
They compared scattering with fluorescence, and they always discussed the same: it is hard to compare since in scattering you don't see small particles and in fluorescence you are not sure whether you are measuring lose QD s or small particles. They always circle back to the idea of looking at data of particles > 50nm, which is a somewhat artificial method.
They also discuss the limitations of nanoparticle tracking analysis , mostly given by not being able to detect particles which are too small or with refractive indexes similar to the medium. What is surprising is that their data changes a lot between fluorescent mode and scattering mode, but they still conclude that they have shown "good concordance".
The main open challenge in using fluorescent labeling for NTA is photobleaching . It is very different in a flow cytometer where you do point detection with a photomultiplier tube than when you do video recordings in wide-field illumination.
Thane, K.E., Davis, A.M. & Hoffman, A.M. Improved methods for fluorescent labeling and detection of single extracellular vesicles using nanoparticle tracking analysis. Sci Rep 9, 12295 (2019). https://doi.org/10.1038/s41598-019-48181-6 ↩