3D Single-Virus Tracking: Advances in Methodology and Labeling Strategies Towards Probing the Virus–Epithelium Interaction

The epithelium represents the first line of defense against viral infection, yet the precise mechanisms by which viruses penetrate this complex barrier remain incompletely understood. Single-virus tracking (SVT) has emerged as a powerful fluorescence microscopy approach to directly visualize viral dynamics with nanometer spatial precision and millisecond temporal resolution. In this review, we survey recent progress in SVT methodologies, from image-based approaches to active feedback techniques, and assess their capacity to resolve viral behavior in physiologically relevant epithelial models. We further evaluate advances in virus labeling strategies—including fluorescent proteins, organic dyes, and nanoparticles—that enable prolonged observation while preserving infectivity. By integrating developments in optical instrumentation and molecular labeling, SVT is increasingly capable of capturing critical processes, including extracellular diffusion, receptor engagement, internalization, and trans-epithelial transport. Finally, we discuss current challenges, including limited penetration depth, photobleaching, and the complexity of 3D epithelial tissues, and outline future opportunities to extend SVT towards in situ and tissue-level studies. Together, these advances position SVT as a transformative tool to illuminate virus–epithelium interactions and guide therapeutic strategies.