Refractive Index and Small Particle Analysis

We often receive requests for silica rather than polystyrene size standards for flow cytometric studies, notably for the analysis of extracellular vesicles. This is because the refractive index (RI) of silica (~1.44) is closer to that of cells (~1.35-1.4) than is the RI of polystyrene (1.59). Polystyrene’s higher RI means greater forward scatter, and as a consequence, polystyrene particles appear to be larger than comparably-sized vesicles.  For this reason, polystyrene nanospheres are not the most faithful surrogates for the establishment of EV size gates. 


So why not use silica spheres in the place of polystyrene? While it may seem like a simple solution, silica cannot be easily dyed.  Enter in the second caveat—the need for fluorescence triggering when analyzing nanoparticles on traditional cytometers. 


All of that said, our fluorescent polystyrene small particle calibration kits and accompanying protocols will permit you to prepare your technicians, instruments and processes for nanoparticle analysis. From strategies for eliminating background particulates that would obscure EVs (flushing and filtration, filtration, filtration!) to fluorescence PMT optimization, our calibration kits and protocols will help to prepare you for the very big job of analyzing very tiny particles. With a bit of work (OK, a fair amount of work), we can get you from here (below left) to there (below right)!  

References