Simultaneous Sizing and Refractive Index Analysis of Heterogeneous Nanoparticle Suspensions

Abstract

Rapid and reliable characterization of heterogeneous nanoparticle suspensions is a key technology across the nanosciences. Although approaches exist for homogeneous samples, they are often unsuitable for polydisperse suspensions, as particles of different sizes and compositions can lead to indistinguishable signals at the detector. Here, we introduce holographic nanoparticle tracking analysis, holoNTA, as a straightforward methodology that decouples size and material refractive index contributions. HoloNTA is applicable to any heterogeneous nanoparticle sample and has the sensitivity to measure the intrinsic heterogeneity of the sample. Specifically, we combined high dynamic range k-space imaging with holographic 3D single-particle tracking. This strategy enables long-term tracking by extending the imaging volume and delivers precise and accurate estimates of both scattering amplitude and diffusion coefficient of individual nanoparticles, from which particle refractive index and hydrodynamic size are determined. We specifically demonstrate, by simulations and experiments, that irrespective of localization uncertainty and size, the sizing sensitivity is improved as our extended detection volume yields considerably longer particle trajectories than previously reported by comparable technologies. As validation, we measured both homogeneous and heterogeneous suspensions of nanoparticles in the 40−250 nm size range and further monitored protein corona formation, where we identified subtle differences between the nanoparticle−protein complexes derived from avidin, bovine serum albumin, and streptavidin. We foresee that our approach will find many applications of both fundamental and applied nature where routine quantification and sizing of nanoparticles are required.

Electrophoretic Deposition Interferometric Scattering Mass Photometry

“…The time varying scattering intensity from a single particle diffusing along a coverslip is not easily modeled. However, this effect might support a nanoparticle tracking analysis strategy that indirectly finds the mass of a particle by measuring its 2D diffusion coefficient , from high frame rate video feed.…”

Characterizing the Backscattered Spectrum of Mie Spheres

“…If one can average over many spheres, some of the methods that have been used are the analysis of the diffuse reflectance and transmittance of particle suspensions, [ 36 ] the refractive index matching of the media surrounding the particles, [ 37 ] the characterization of the light reflected off the Bragg planes of spherical particle clusters, [ 38,39 ] or holographic microscopy applied to a polydisperse nanoparticle suspension. [ 40 ] Nonetheless, these methods cannot be used for the characterization of a single isolated particle. This can be done by using holographic techniques, [ 41 ] or by means of an optical trapping setup by fitting the dynamic behavior of a spherical particle [ 42 ] or a biological sample.…”

Size photometry and fluorescence imaging of immobilized immersed extracellular vesicles

“…1a. We use the terminology 'size photometry' because nanoparticles are conventionally characterized by size, however, we note that iSCAT contrasts are principally dependent on both EV size and refractive index and only few implementations truly decouple them [25][26][27] . Throughout this manuscript, we validate the SP sizing accuracy for multiple types of large nanoparticles with increasing complexity (various solid nanoparticles, liposomes, and EVs).…”

“…1a. We use the terminology 'size photometry' 6 because nanoparticles are conventionally characterized by size, however, we note that iSCAT contrasts are principally dependent on both EV size and refractive index and only few implementations truly decouple them [25][26][27] . A detailed description of the iSCAT imaging principle is given in Supplementary Section S1, describing the mathematical underpinning of its increased sensitivity for small particles compared to darkfield imaging.…”