The development of single virus tracking (SVT) methods has given insight into previously unresolved phases in viral infection. SVT methods have revealed critical steps in viral internalization, envelope fusion, endocytic trafficking, and even virion assembly, but these studies share a similar shortcoming. Current live-cell virus tracking methods can not probe deep into samples and mainly capture dynamics near the coverslip surface, an interface foreign to the cell that may not reflect actual in vivo behavior. Over the prior funding period, we developed a powerful SVT microscopy technique for high-speed 3D tracking of single viruses. This method, called 3D Tracking and Imaging (3D-TrIm), uses active feedback to lock on to the real-time position of freely diffusing virions (up to 10 µm2/s) with high temporal resolution (down to 10 µsec) deep within samples (axial ranges up to 50 µm). Critically, 3D-TrIm simultaneously captures volumetric images of the cellular environment, placing these high-speed viral dynamics in context. Using 3D-TrIm, we seek to delve far beyond the coverslip to perform single virus tracking in the complex environments where viral infection truly occurs: the epithelia. The epithelia, be it in the lung or the gastrointestinal tract, is a complex tissue with an extended mucus layer and glycocalyx. These act as an important barrier to viral and bacterial infection and are entirely missing in studies on simple cultured cells. Initial experiments will focus on how the surface of cultured cells affects and inhibits the dynamics of freely diffusing VSV-G and SARS-CoV-2 pseudotyped virus-like particles. High-speed 3D single virus tracking will then be performed in real epithelial cultures to provide a first glimpse at the dynamics of single viruses navigating through the viscous mucus layer and the size-excluding periciliary layer. The proposed work will also probe how influenza A (IAV) uses a balance of receptor-binding (hemagglutinin) and receptor-destroying (neuraminidase) envelope proteins to get through the native epithelia. We will test the hypothesis that IAV acts as a "molecular walker" using a Brownian ratchet mechanism to process through the epithelia. The validity of the "molecular walker" model will be further probed by tracking HA- and NA-functionalized silver nanoparticles with nm spatial and µsec temporal resolution to look for processivity along sialic acid residues. Finally, we aim to advance 3D-TrIm towards tracking in more complex tissue environments. Tracking in highly scattering and highly autofluorescent tissues will be accomplished with recursive Bayesian filtering and information-efficient sampling to distinguish photons from tracked particles versus background photons. The studies proposed herein will be the first to probe viral dynamics in the complex epithelia. The dynamics of the tracked viral particles will yield critical insight into the mechanism of viral infection through a host organism's first line of ...