Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light

A quadrant photodiode placed in the back-focal plane of the microscope of a laser trap provides a high-resolution position sensor. We show that in add ition to the lateral displacement of a trapped sphere, its axial position c an be measured by the ratio of the intensity of scattered laser light; to t he total amount of the light reaching the detector. The addition of the axi al information offers true three-dimensional position detection in solution , creating, together with a position control, a photonic force microscope w ith nanometer spatial and microsecond temporal resolution. The measured pos ition signals are explained as interference of the unscattered trapping las er beam with the laser light scattered by the trapped bead. Our model expla ins experimental data for trapped particles in the Rayleigh regime (radius a < 0.2 lambda) for displacements up to the focal dimensions. The cross-tal k between the signals in the three directions is explained and it is shown that this cross-talk can be neglected for lateral displacements smaller tha n 75 nm and axial displacements below 150 nm. The advantages-of three-dimen sional single-particle tracking over conventional video-tracking are shown through the example of the diffusion of the GFI-anchored membrane protein T hy1.1 on a neurite. (C) 1999 Wiley-Liss. Inc.