Enriched Spatiotemporal Mapping of Single-Virus Dynamics in Cellular and Complex Environment via High-Speed 3D Tracking Microscopy
Viral infection is a highly dynamic process spanning molecular to tissue scales, posing persistent challenges for real-time, high-resolution imaging. This dissertation presents advancements in high-speed 3D tracking microscopy and photostable labeling to enable enriched spatiotemporal mapping of single-virus dynamics in both cellular and complex environments. We developed 3D-TrIm microscopy, combining active-feedback single-particle tracking with simultaneous volumetric imaging, to resolve viral behaviors such as membrane skimming, receptor binding, and intercellular trafficking in live cells and tissue-like systems. To extend tracking duration, photostable StayGold-labelled virus-like particles were engineered, achieving continuous, kilohertz-rate 3D tracking for over an hour. Integrating these tools, we investigated SARS-CoV-2 infection dynamics and uncovered a previously unreported actin-dependent viral membrane trafficking behavior associated with ACE2 expression. Finally, we applied single particle tracking microscopy to study nanoparticle diffusion in 3D porous hydrogels, revealing heterogeneous transport modes and confinement dynamics, offering insights into material microstructure and particle transport relevant to biological and biomedical applications.