Virion Dynamics in the Extracellular Space Explored by 3D Tracking and Imaging Microscopy

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2022

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Abstract

The early stages of the virus-cell interaction have long evaded observation by existing microscopy methods due to the rapid diffusion of virions in the extracellular space and the large 3D cellular structures involved. Here we present an active-feedback single-virus tracking method with simultaneous volumetric imaging of the live cell environment to address this knowledge gap to present unprecedented detail to the extracellular phase of the infectious cycle. Additionally, we report the development of a solution for rapid imaging using a tessellating scan pattern that can be used as a novel way to perform rapid 3D imaging at up to 8x faster than conventional z-stack method. This new method is also capable of imaging with a moving stage, making it the ideal compliment to active-feedback tracking systems. We report previously unobserved phenomena in the early stages of the virus-cell interaction, including skimming contact events at the millisecond timescale, orders of magnitude change in diffusion coefficient upon binding, and cylindrical and linear diffusion modes along filopodia. Finally, we demonstrate how this new method can move single-virus tracking from simple monolayer culture towards more tissue-like conditions by tracking single virions in tightly packed epithelial cells. This multi-resolution method presents new opportunities for capturing fast, 3D processes in biological systems.

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Johnson, Courtney C (2022). Virion Dynamics in the Extracellular Space Explored by 3D Tracking and Imaging Microscopy. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/25275.

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