Size distribution measurement of polydisperse macromolecular samples using nanoparticle tracking analysis

The standard technique used to measure the size distribution of nanometer-sized particles in suspension is dynamic light scattering (DLS). Recently, nanoparticle tracking analysis (NTA) has been introduced to measure the diffusion coefficient of particles in a sample to determine their size distribution in relation to DLS results. Because DLS and NTA use identical physical characteristics to determine particle size but differ in the weighting of the distribution, NTA can be a good verification tool for DLS and vice versa. In this study, two NTA data analysis methods based on maximum-likelihood estimation were evaluated, namely finite track length adjustment and an iterative method, on monodisperse polystyrene beads and polydisperse vesicles by comparing the results with DLS. The NTA results from both methods agreed well with the mean size and relative variance values from DLS for monodisperse polystyrene standards. However, for the lipid vesicles prepared in various polydispersity conditions, the iterative method resulted in a better match with DLS. Further, it was found that it is better to compare the native number-weighted NTA distribution with DLS, rather than its converted distribution weighted by intensity. Nanoparticle tracking analysis is a size measurement technique that determines the size distribution of particles in suspension by tracking individual particles undergoing Brownian motion. A key element in the measurement analysis is the recognition radius, which distinguishes the individual, tracked particles from one another. However, by defining a finite radius, the displacement of tracked particles is effectively restricted, translating into an overestimation of particle size. A modified probability model that describes the restricted displacement of a tracked particle is introduced to achieve more accurate size distribution determination. Through virtual NTA measurement by computer simulations and real NTA experiments, the analytical performance of the modified displacement probability was tested in comparison to the conventional probability. Whereas the conventional displacement probability results in an overestimation of the particle size, the modified displacement probability mitigates the effect of the overestimation and provides more accurate mean size within an error of less than 6% the nominal size.Doctor of Philosoph