3D 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. The 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 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. The current system has a frequency response at the video rate and an axial range of 100 μm. We tested the system by tracking well-defined test trajectories, and by tracking particles in model systems. 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