Enriched Spatiotemporal Mapping of Single-Virus Dynamics in Cellular and Complex Environments via High-Speed 3D Tracking Microscopy

Abstract

Viral infection are dynamic processes, covering vast spatiotemporal scales within heterogeneous biological environments. Extracellular viral diffusion features high diffusivity (~ 2 μm2/sec) across large axial range. Virus-receptor binding happens in milliseconds on a molecular scale while the whole infection cycle lasts from hours to days in cellular to tissue level. Capturing these events in real time with high spatiotemporal resolution has remained a persistent challenge due to limitations in conventional microscopy techniques. This dissertation presents a suite of innovations in high-speed 3D tracking microscopy and labelling strategies aimed at enabling enriched spatiotemporal mapping of single-virus dynamics in both cellular and complex environments. To overcome the limitations of existing single virus tracking (SVT) methods, we developed 3D tracking and imaging (3D-TrIm) microscopy, an active-feedback single-particle tracking platform (3D-SMART) integrated with simultaneous volumetric live-cell imaging (3D-FASTR). This technique offers unprecedented resolution of the extracellular phase of viral infection, visualizing insightful viral infection events such as membrane skimming, receptor binding, viral trafficking along protrusion, and intercellular trafficking. Furthermore, 3D-TrIm is capable of extending SVT beyond simple monolayer cell culture to more complex tissue-like epithelial systems, enabling comprehensive mapping of viral behavior within more biological relevant environments. Despite the performance of 3D-TrIm, SVT trajectories typically only last for minutes due to photobleaching of fluorescent labels, failing to visualize the entire virus infection cycle. Recognizing the need for photostable and bright fluorescent labels to sustain long-duration high-speed tracking, we incorporated StayGold, a highly photostable green fluorescent protein, to generate virus-like particles (StayGold-VLPs). These newly engineered particles enabled continuous 3D SVT in live cells for over an hour while maintaining the spatial resolution and kilohertz sampling rates, dramatically increasing the photon budget and information content of individual trajectories. This enhanced capability facilitates detailed studies of viral trafficking dynamics over extended timescales and offers the potential to map the complete life cycle of a single virus in the future. Previous studies have demonstrated the capability of 3D-TrIm microscopy to perform single virus tracking with high spatiotemporal resolution, as well as the effectiveness of StayGold-based VLP labelling strategy in extending tracking duration. We combined these two advancements to investigate the viral invasion dynamics of SARS-CoV-2. Long-term and eventful infection trajectories were captured, including free diffusion, viral landing, diffusion on cellular protrusion, internalization and internal trafficking. Among those events, a novel diffusion pattern was observed: viral linear trafficking on plasma membrane, which helps the cell to explore the cellular surface rapidly to localize the optimal entry spot. Further study revealed that this trafficking pattern is actin-dependent and positively associated with the ACE2 expression. Finally, we applied these high-speed 3D tracking methodologies to investigate nanoparticle diffusion in 3D porous environments. Using 3D-SMART microscopy, we resolved nanoparticle dynamics within agarose hydrogels at super resolution (~ 10 nm in XY, ~ 30 nm in Z). These experiments uncovered various transport behaviors, highlighting here ‘hopping diffusion’, where particles intermittently escape confinement pockets, providing insights into hydrogel microstructures. Long, highly sampled trajectories enable extraction of kinetic parameters, confinement structure and size, and thermodynamic barriers, offering new perspectives on nanoparticle diffusion and the structural dynamics of porous materials, with implications for drug delivery, material science, and biological systems.