Characterization of Nanoparticles in Suspension by Simultaneous iNTA and Fluorescence Detection with Single-Molecule Sensitivity

Quantitative knowledge of nanoparticle properties is desirable in a large number of scientific and technological applications, but measurements with a high degree of precision usually prove to be challenging. Among a range of available methodologies, optical techniques with single-particle sensitivity are especially interesting because they can reveal intrinsic heterogeneities in a fast, noninvasive manner. Recently, we presented interferometric nanoparticle tracking analysis (iNTA) as a highly sensitive label-free technique that is capable of determining the size, concentration, and index of refraction of different subpopulations in a suspension mixture. Here, we enhance this method with biochemical specificity through multicolor fluorescence detection at the single-molecule sensitivity limit. We benchmark the performance of the combined technique, which we name iNTA-F, by distinguishing populations of fluorescent and nonfluorescent nanoparticles of different material, size, and fluorescence intensity, with an emphasis on the characterization of lipid vesicles and biological extracellular vesicles (EVs).