High Resolution Image Download MS PowerPoint Slide We report the first application of three-dimensional (3D) single-particle tracking (SPT) to hydrodynamic size characterization of gold nanoparticles in water. Nanoparticles undergoing Brownian motion were dynamically locked at the focal point of a microscope objective, one at a time, by rapid counteractive movements of the sample container. The hydrodynamic radius was derived from the recorded trajectory of each individual nanoparticle. The directly measured size and size distribution using 3D-SPT were in agreement with those obtained using the conventional dynamic light scattering (DLS) and using transmission electron microscopy (TEM), respectively.