Three dimensional single-particle tracking with nanometer resolution

Abstract

We have developed a method for three dimensional (3D) tracking of polystyrene spheres with nanometer resolution. The detection technique is based on measuring the displacement of a polystyrene sphere positioned in the center of a laser beam just behind the focus. A change in the lateral position of the sphere causes a deflection of the beam which can be measured using a position sensitive detector. A change in the axial position of the sphere causes a shift in the axial position of the focus behind another lens, which can be measured using an overfilled photodiode. A feedback system is used to keep the sphere in the center of the laser beam to avoid the influence of lateral displacements on the detection of the axial position. Spatial resolution for a 0.92 μm polystyrene sphere was better than 1 nm in three dimensions using a sampling rate of 1 kHz. This method was applied to track spheres bound to adhesion molecules LFA-1 expressed at the surface of living cells. It turned out to be a useful method to accurately measure the 3D trajectory of biological molecules on cells in real time.