Three-dimensional single particle tracking on the two-photon microscope

We have developed a 3D single-particle-tracking (SPT) system based around the two-photon laser-scanning fluorescence microscope that can track particles in all three dimensions and at a high frequency response. We have implemented two different techniques to achieve this goal. Both techniques employ feedback control in order to track the particle but differ in the approach they use to ascertain the particle's 3D position. The first technique scans a small volume around a particle to build up a volumetric image that is then used to determine the particle's position. The second technique scans only a single plane but utilizes optical aberrations which have been introduced into the optical system that break the axial symmetry of the point spread function and serves as an indicator of the particle's axial position. We identified several different modes of motion in sucrose solutions and agarose gels, including the transient trapping of particles in the microdomains of agarose gels. As an application of this technique, we have investigated the viscoelastic mechanical response of biological cells. The study involves attaching magnetic spheres to the surface of a cell, and then applying a magnetic field and monitoring the response of nearby fluorescent polystyrene spheres. We have observed significant motion of the cell in all three dimensions and have strong evidence that in order to adequately model the mechanical response of the cell it is important to monitor the motion in all three dimensions.