Extracellular vesicles (EVs) are nanoparticles released by cells and have high potential as disease biomarkers. EVs are studied by flow cytometers, which measure scattering in arbitrary units. With Mie theory, arbitrary units can be related to diameter when the particle refractive index (RI) is known. However, a setup to determine the RI of nanoparticles in a traceable manner, including uncertainty, is lacking. Therefore, we have developed the first metrological flow cytometer, utilizing Laguerre-Gauss illumination, multi-angle light scattering, artificial intelligence data processing, and a calibrated syringe pump, to traceably determine the size, RI, and concentration of single nanoparticles in liquid.
Refractive index (RI) measurements are highly dependent on environmental conditions, which are often not reported, resulting in non-traceable measurements. Additionally, the RI of buffer solutions, such as phosphate-buffered saline (PBS), is unknown. We built a new optical set-up based on the minimum deviation angle to traceably measure the RI of solids and liquids under controlled environmental conditions. We measured the RI of fused silica, 1.470091, and PBS, 1.344599, at 405 nm, 20.00 °C with an expanded uncertainty of 1.4e-6. Our results differ from previously assumed RIs and therefore have practical implications for nanoparticle flow cytometry measurements.
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