To better understand how viruses breach the protective layers of cells that line the airways and gut, Duke University researchers used multimodal microscopy to capture real-time video footage of viruses as they approach their cellular targets. Using the technique, called 3D Tracking and Imaging Microscopy (3D-TrIm), the researchers observed previously unobserved phenomena in the early stages of the virus-cell interaction, including skimming contact events at the millisecond timescale. According to professor Kevin Welsher, the critical moments before an infection begins have been difficult to view with existing microscopy methods. One reason is that viruses move two to three orders of magnitude faster in the unconfined space outside the cell, compared with its crowded interior. In addition, viruses present an imaging challenge because they are hundreds of times smaller than the cells they infect. The multimodal 3D-TrIm instrument integrates a real-time, active-feedback, single-particle tracking method with simultaneous volumetric imaging of the live cell environment. The instrument provided the researchers with a glimpse of the initial moments of the virus-cell interaction that they would not be able to view using the independent components of the instrument alone. In the future, the researchers hope that the technique could be used to observe viruses in real time in conditions that imitate the environment in which an infection first takes hold. The multimodal method essentially combines two microscopes in one. The first microscope locks on to the virus. To calculate and update its position, the microscope sweeps a laser around the fast-moving virus tens of thousands of times per second. The microscope stage continuously adjusts itself to maintain focus on the virus as it moves around the exterior of the cell. A fluorescent label is attached to the virus to enable tracking. While the first microscope tracks the virus, the second microscope takes images of the surrounding cells. The combined effect is similar to navigating in a car with Google Maps. Comparable to the way one’s physical location within a larger context is shown in Google Maps, the current location of the virus and its surrounding environment is shown simultaneously in 3D-TrIm, only it is shown in 3D. Using 3D-TrIm, the team acquired over 3000 viral trajectories and demonstrated how 3D-TrIm’s use of localizations in time is comparable to superresolution microscopy methods. The “supertemporal resolution” provided by 3D-TrIm allowed trajectories to be formed and changes in diffusive regimes to be detected. It also made it possible to trace structural features at the nanoscale. The researchers also used 3D-TrIm to track single viruses in tightly packed epithelial cells, to demonstrate how 3D-TrIm could advance single-particle tracking from simple monolayer cell culture to more realistic, tissue-like conditions. To the best of the team’s knowledge, the rapid diffusion of single viruses in a tightly packed epithelial layer has not been reported until now. Currently, the researchers can only track a virus for a few minutes at a time before the fluorescent label goes dim. “The biggest challenge for us now is to produce brighter viruses,” researcher Jack Exell said. To help 3D-TrIm to correlate extracellular and cell-surface dynamics with the infectivity of a single virus, the researchers developed an information-efficient sampling approach that extends the observation time by only sampling high-information areas around the virus particle. The researchers believe that their approach could be extended to the study of pseudotyped SARS-CoV-2 viruses, as well as to any system where fast dynamics of nanoscale objects occur over large volumetric scales, including the delivery of nanoscale drug candidates. The research was published in Nature Methods (www.doi.org/10.1038/s41592-022-01672-3).