Three-Dimensional Tracking of Tethered Particles for Probing Nanometer-Scale Single-Molecule Dynamics Using a Plasmonic Microscope

Three-dimensional (3D) tracking of surface-tethered single particles reveals the dynamics of the molecular tether. However, most 3D tracking techniques lack precision, especially in the axial direction, for measuring the dynamics of biomolecules with a spatial scale of several nanometers. Here, we present a plasmonic imaging technique that can track the motion of ∼100 tethered particles in 3D simultaneously with sub-nanometer axial precision and single-digit nanometer lateral precision at millisecond time resolution. By tracking the 3D coordinates of a tethered particle with high spatial resolution, we are able to determine the dynamics of single short DNA and study its interaction with enzymes. We further show that the particle motion pattern can be used to identify specific and nonspecific interactions in immunoassays. We anticipate that our 3D tracking technique can contribute to the understanding of molecular dynamics and interactions at the single-molecule level.