Research

Introduction to 3D real-time single-particle tracking

Many cell-biology measurements suffer from two limitations. One limitation is speed. Conventional confocal microscopy builds images by scanning point-by-point, which is too slow for fast moving objects. Another limitation is the lack of ability to reveal the heterogeneity of busy and chaotic intracellular worlds. Many common experiments aggregate information from thousands of molecules at once and report a statistical observation. This can blur rare or transient events - the moments when a single molecule changes state, binds and escapes, or a virus enters a host cell.

My work aims to address these problems by developing 3D Real-time Single-particle Tracking (3D-RT-SPT) microscopes that can continuously chase a single particle or molecule rather than repeatedly imaging the whole scene. We added high-speed optics - dual electro-optic deflectors (EODs) for latent scanning and a TAG lens for axial scanning - to sweep a focused laser spot around the particle. From the resulting photon stream, a control algorithm estimates the particle's 3D offset and guides a piezo stage to keep the particle centered. This system, we call 3D Single Molecule Active Real-time Tracking (3D-SMART) microscopy, produces continuous 3D trajectories with rich, time-resolved photon data, making it possible to study single-molecule dynamics at the timescales where biology actually happens.

The system was originally introduced in Shangguo's 2017 Optics Letters paper and later optimized in the 2020 Nature Communications paper.