Capturing the start point of the virus–cell interaction with high-speed 3D single-virus tracking

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 three-dimensional cellular structures involved. Here we present an active-feedback single-particle tracking method with simultaneous volumetric imaging of the live cell environment called 3D-TrIm to address this knowledge gap. 3D-TrIm captures the extracellular phase of the infectious cycle in what we believe is unprecedented detail. We report what are, to our knowledge, 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 cellular protrusions. Finally, we demonstrate how this method can move single-particle tracking from simple monolayer culture toward more tissue-like conditions by tracking single virions in tightly packed epithelial cells. This multiresolution method presents opportunities for capturing fast, three-dimensional processes in biological systems.

Main

The ongoing SARS-CoV-2 pandemic has demonstrated with frightening clarity the need for fundamental research in physical virology to exploit and counter the mechanisms of viral infection. The first point of contact with the host organism occurs in the extracellular space of the epithelia, whose cells form a tightly packed arrangement protected by an extended mucus layer and glycocalyx^1,2,3,4,5. The structure of the extracellular matrix (ECM) has been shown to be critical to the viral lifecycle, undergoing changes in structure and composition upon introduction of viral pathogens⁶ and hosting biofilm-like viral assemblies for cell-to-cell transmission⁷.

Single-particle tracking (SPT) methods have emerged as a powerful tool in our understanding of viral infection^8,9,10,11,12. These methods have uncovered virion binding mechanisms to the cell surface^13,14, distinguished internalization pathways^{15,16,17,18,19}, identified the cellular location of envelope fusion^{20,21,22,23,24,25,26}, characterized cytoskeletal trafficking^{14,19,20,21,22,27,28,29,30,31,32,33} and demonstrated how viruses hijack filopodia to efficiently infect neighboring cells^{19,23,34,35,36,37,38,39}.

Despite these advances, it has thus far not been possible to follow virions in the extracellular space with sufficient detail to interrogate this important phase of viral infection. This is because extracellular diffusion occurs across depth ranges exceeding 10 μm and at diffusive speeds 2–3 orders of magnitude greater than the highly confined processes that can be followed by conventional SPT methods. Even the most advanced image-based SPT methods such as spinning-disk confocal (SDC) and light-sheet microscopy^40,41 are unable to meet these challenges as they suffer from limitations caused by attempting to simultaneously track and image disparately scaled objects on a single platform.

One way to understand these limitations is to consider the sampling of trajectory points in terms of the number of localizations per second (loc s⁻¹), which for image-based tracking methods is given by the volumetric imaging rate (Fig. 1a). As volumetric data are acquired frame by frame, the temporal resolution scales poorly as the axial extent of the process in question grows. Yet shrinking the volume Z size reduces the likelihood that the virion will remain in the volumetric field of view, shortening the observation duration and, thus, the absolute number of localizations acquired. Additionally, as camera exposure times decrease, photon limitations when trying to image single particles become a limiting factor. Given a modest value of 16 z-slices spaced over an axial range sufficient to capture extracellular diffusion, even the fastest light-sheet systems can theoretically acquire as many as 6.25 loc s⁻¹ at best. Overcoming these fundamental limitations in speed and axial range motivated the development of active-feedback tracking methods which focus exclusively on the tracked particle⁴². Such methods can localize particles with high spatial and temporal resolution, but the cost to obtaining this enhanced temporal resolution is loss of environmental context. While such methods have been useful in studying particle dynamics in isolation, this missing context has precluded the application of such methods to studying particle–environment interactions.

To address this gap, we present 3D Tracking and Imaging Microscopy (3D-TrIm), a multi-modal instrument that integrates a real-time active-feedback tracking microscope^43,44,45 with a volumetric imaging system⁴⁶ capable of simultaneously tracking the high-speed dynamics of extracellular virions while imaging the surrounding three-dimensional (3D), live cell environment (Fig. 1a). This multi-modal approach provides unique value which cannot be extracted by its independent components alone and, as demonstrated here, provides unprecedented glimpses into the initial moments of the virus–cell interaction. Using 3D-TrIm, we acquired over 3,000 viral trajectories and demonstrate the benefits of how this contextualized tracking uses localizations in time analogously to super-resolution methods. This ‘super-temporal resolution’ enables not just the formation of trajectories, but the detection of changes in diffusive regimes and the tracing out of nanoscale structural features. Finally, 3D-TrIm was applied to advance SPT from monolayer cell culture to tightly packed, 3D epithelial culture models, providing a window into this critical stage of viral infection.

Results

Capturing extracellular viral dynamics requires the ability to track a rapidly diffusing target in three dimensions. To accomplish this, we apply the recently developed 3D single-molecule active real-time tracking (3D-SMART)^43,44. 3D-SMART is an active-feedback microscopy method that uses real-time position information to ‘lock on’ to moving fluorescent targets. Critically, 3D-SMART can capture particles diffusing at up to 10 μm² s⁻¹ with only a single fluorophore label⁴³, making it an ideal choice for capturing diffusing virions⁴⁵.