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Radial profiles, b( r), are obtained from these holograms and their fits by averaging the normalized intensity over angles around the center of each feature and are plotted as a function of distance r from the center of the feature. The fit to each hologram yields values for the particle’s radius, a p, and refractive index, n p. These typical examples were obtained for spheres with radii a p = 0.237 μm (224 pixel × 224 pixel region of interest), 0.800 μm (356 pixel × 356 pixel), and 10.47 μm (608 pixel × 608 pixel). (b) Measured holograms of colloidal polystyrene spheres in water together with fits, demonstrating the range of particle sizes amenable to holographic characterization. These results establish holographic characterization’s ability to differentiate particles by composition as well as by size. Superimposed crosses indicate the manufacturer’s specification for each of the 4 populations. Results for 20,000 particles are plotted. (a) Scatter plot of radius a p and refractive index n p obtained with holographic characterization of the 4-component stoichiometric colloidal mixture described in the Materials section.

Holographic characterization measurements are compared with results obtained with microflow imaging and dynamic light scattering.īiopharmaceuticals characterization colloids light scattering microparticles microscopy particle size protein aggregation.Ĭopyright © 2016 American Pharmacists Association®. Differentiation is demonstrated with samples that have been spiked with separately characterized silicone spheres. These capabilities are demonstrated through measurements on samples of bovine pancreas insulin aggregated through centrifugation and of bovine serum albumin aggregated by complexation with a polyelectrolyte. Information on individual particle's refractive indexes can be used to differentiate protein aggregates from such contaminants as silicone droplets. The measurement proceeds fast enough to build up population averages for time-resolved studies and lends itself to tracking trends in protein aggregation arising from changing environmental factors. Holographic characterization directly measures the radius and refractive index of subvisible protein aggregates and offers insights into their morphologies. We demonstrate how holographic video microscopy can be used to detect, count, and characterize individual micrometer-scale protein aggregates as they flow down a microfluidic channel in their native buffer.