Real-time 3D single-molecule tracking
The measurement of dynamic biological processes in live cells has been fundamentally limited by slow data acquisition in three dimensions. Our aim is to develop new microscopy methods to enable real-time observation of fast 3D molecular phenomena in their native biological context. Our approach is to use active feedback or real-time 3D single-particle tracking to enable high spatiotemporal resolution measurements of single nanoscale objects undergoing rapid 3D diffusion. Using real-time position measurement and high-speed sample positioning, a freely diffusing object can be “locked” relative to the objective focal volume, allowing continuous high-speed observation on rapidly evolving processes. We are actively pushing the limits of this active feedback technology to higher speeds to achieve real-time tracking on freely diffusing single molecules.
Movie 1: Real-time 3D tracking of freely diffusing 100 nm nanoparticles using 3D Dynamic Photon Localization Tracking (3D-DyPLoT). A single freely diffusing particle is “locked” in the focus of the objective lens using a fast piezoelectric stage and an optical feedback loop.
Selected Publications
Tan, X. & Welsher, K. Particle-by-Particle in Situ Characterization of the Protein Corona via Real-Time 3D Single Particle Tracking Microscopy. Angewandte Chemie International Edition. (2021). Forthcoming. (chemrxiv).