Precision size and refractive index analysis of weakly scattering nanoparticles in polydispersions

Abstract

Characterization of the size and material properties of particles in liquid suspensions is in very high demand, e.g., for the analysis of colloidal samples or of bodily fluids such as urine or blood plasma. However, the existing methods are limited in deciphering the constituents of realistic samples. Here, we introduce iNTA as a new method, which combines interferometric detection of scattering with nanoparticle tracking analysis, to reach an unprecedented sensitivity and precision in determining the size and refractive index distributions of nanoparticles in suspensions. After benchmarking iNTA with samples of colloidal gold, we present its remarkable ability to resolve the constituents of various multi-component and polydisperse samples of known origin. Furthermore, we showcase the method by elucidating the refractive index and size distributions of extracellular vesicles from Leishmania parasites and nanoparticles in human urine. The current performance of iNTA already enables advances in several important applications, but we also discuss possible improvements.

Size photometry and fluorescence imaging of immobilized immersed extracellular vesicles

“…FCM and NTA lines represent the commercial instruments used in this manuscript, whereas the dashed FCM line indicates the highest reported sensitivity on any FCM to our knowledge 9 . Note that NTA and iNTA 25 report hydrodynamic diameters which are different to the physical EV size reported by SP and FCM.…”

“…6. The SP setup itself is similar to iNTA 25,26 and in-solution measurements of absolute EV concentrations and hydrodynamic diameters are possible but will require more powerful illumination and high speed cameras to achieve similar LODs to SP. We propose the use of SP with fluorescence imaging as in SPFI as the main application which replicates the defining features of flow cytometry that can simultaneously capture fluorescence and label-free side scattering data.…”

“…3c), we observed a contrast inversion under 440 nm illumination which requires special attention for SP sizing. The standard mathematical model of interferometric imaging (Supplementary Section 1) predicts that contrasts 𝐶 initially scale with particle diameters 𝑑 to the third power 25 . This 𝐶 ∝ 𝑑 " scaling is an approximation that breaks for large particles, requiring experimental validation to define its range of applicability and establish the maximum sizing range achievable with SP.…”

“…S2a) and FEM simulations (Fig. S7), where we compared EVs modeled with solid sphere equivalent refractive index to core-shell models 25,34 in which membrane and lumen contribute independently to EV scattering signals.…”

Precise and Dynamic Temperature Control in High-Resolution Microscopy with VAHEAT

“…A well-controlled temperature in the FOV is markedly important in order to extract and model systemrelevant parameters. In a recent preprint, Kashkanova et al [15] developed a method based on interferometric scattering (iSCAT) microscopy to evaluate complex mixtures of nanoparticles. Our device was used to characterize temperature dependence of diffusion constant of gold nanoparticles.…”