Real-time 3D single-particle tracking has emerged as a powerful tool for investigating dynamic biological interactions and single-molecule tracking has the potential to revolutionize the study of biological systems due to their high spatial and temporal resolution and high sensitivity. Here I will discuss several 3D tracking methods developed by our lab. (i) 3D Dynamic Photon Localization Tracking (3D-DyPLoT). 3D-DyPLoT dynamically scans a focused laser spot in 3D with a tunable acoustic gradient lens and a 2D electro-optic deflector and has demonstrated real-time tracking at high diffusive speeds (up to 20 µm2/s) and low photon count rates 10 kHz. (ii) 3D Precision Adaptive Real-time Tracking (3D-PART). 3D-PART can trade tracking speed for precision on demand in real time and capture the heterogeneous dynamics. (iii) 3D Single-Molecule Active Real-time Tracking (3D-SMART). 3D-SMART can continuously track single fluorescent dye molecules for several minutes at a time, two orders of magnitudes longer than previously reported methods. We demonstrated the application of this new single-molecule tracking microscopy to track the free, 3D diffusion of DNA, RNA and proteins in solution. 3D-SMART was also applied to observing the DNA transcription events in solution. This real-time 3D single molecule tracking method promises to be a powerful tool for capturing the dynamics of single biomolecules at high speeds and over 3D distances. Shangguo Hou is a 5th postdoctoral researcher.