This video is adapted from 10.3390/e23050498
3D Real-Time Single Particle Tracking (3D-RT-SPT) has greatly expanded our knowledge in sophisticated intracellular events. Various sampling patterns were implemented in different 3D-SPT methodologies for real-time estimation of particle positions. However, information efficiency of different sampling patterns was rarely investigated experimentally. It is extremely important to extract more information from each photon as many emitters used in living systems undergo irreversible photobleaching after giving out a limited number of photons. In this video we demonstrate how we systematically investigated photon efficiency of different sampling patterns in the XY-plane, along the Z-axis and in 3D in our highly tunable 3D Single Molecule Active Real-time Tracking (3D-SMART) system. We show that information-efficient patterns are off-centered, overlapped with high Fisher information (FI) areas. In the XY-plane or along the Z-axis, doubled precision was obtained and a 15% increase in precision was obtained in 3D. By adjusting sampling density in 3D, it was also possible to obtain unbiased, isotropic precision in 3D despite different size of PSF scale in the XY-plane and along the Z-axis. As FI can be experimentally determined by evaluating the point-spread function of specific emitters and sampling patterns can be designed accordingly, the findings of this research can be easily applied to different systems to achieve optimal information efficiency.