We have developed a method for three dimensional (3D) tracking of poly
styrene 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 c
an be measured using a position sensitive detector. A change in the ax
ial 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 mu
m 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 traj
ectory of biological molecules on cells in real time. (C) 1998 America
n Institute of Physics. [S0034-6748(98)01607-4].