Fluorescent Labeling of Extracellular Vesicles

<p> Characterizing <a class="wikilink" href="/ev/"> EV </a> s is of paramount interest for people thinking in diagnostic applications. Measuring the <a class="wikilink" href="/size_of_nanoparticles/"> size of nanoparticles </a> is, of course, one of the features that people look into, the other are the specific <a class="wikilink" href="/phenotypes/"> phenotypes </a> of the vesicles. For this, <a class="wikilink" href="/fluorescence/"> fluorescence </a> is an ideal tool, because it can target specific proteins within a distribution of particles. The key objective is to study <a class="wikilink" href="/phenotype/"> phenotype </a> or <a class="wikilink" href="/epitope/"> epitope </a> prevalence. </p> <p> A by-product of <a class="wikilink" href="/fluorescent_labeling/"> fluorescent labeling </a> is that <a class="wikilink" href="/nanoparticle_tracking_analysis/"> nanoparticle tracking analysis </a> 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 <sup id="fnref:1"> <a class="footnote-ref" href="#fn:1"> 1 </a> </sup> developed a protocol to study the effect that labeling with <a class="wikilink" href="/quantum_dots/"> quantum dots </a> has on the final result. They also looked into how some purification strategies alter the results. </p> <p> 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 <a class="wikilink" href="/qd/"> QD </a> s or small particles. They always circle back to the idea of looking at data of particles &gt; 50nm, which is a somewhat artificial method. </p> <p> They also discuss the <a class="wikilink" href="/limitations_of_nanoparticle_tracking_analysis/"> limitations of nanoparticle tracking analysis </a> , mostly given by not being able to detect particles which are <em> too small </em> 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". </p> <p> The main open challenge in using fluorescent labeling for <a class="wikilink" href="/nta/"> NTA </a> is <a class="wikilink" href="/photobleaching/"> photobleaching </a> . It is very different in a <a class="wikilink" href="/flow_cytometer/"> flow cytometer </a> where you do point detection with a <a class="wikilink" href="/photomultiplier_tube/"> photomultiplier tube </a> than when you do video recordings in wide-field illumination. </p> <div class="footnote"> <hr/> <ol> <li id="fn:1"> <p> Thane, K.E., Davis, A.M. &amp; 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 <a class="footnote-backref" href="#fnref:1" title="Jump back to footnote 1 in the text"> ↩ </a> </p> </li> </ol> </div>