Yuxin Lin

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.

Early interactions between viruses and live cells are difficult to resolve due to rapid extracellular motion, 3D nature of the cell membrane, and the fast, nanoscale interactions involved. While actin is a central regulator of viral entry, direct observations of actin-aided trafficking have been restricted to membrane protrusions on glass surfaces given the limitations of conventional methods. Here, high-speed 3D Tracking and Imaging microscopy (3D-TrIm) is integrated with highly photostable StayGold-labeled SARS-CoV-2 virus-like particles to capture long-term, high-resolution single-virus trajectories in live cells. This approach revealed distinct regimes of viral dynamics, including extracellular diffusion, protrusion-based surfing, and an unreported linear trafficking mode along the plasma membrane that precedes viral internalization. This work demonstrates that this membrane trafficking is actin-driven and positively correlated with ACE2 expression. These findings reveal new actin exploitation by viruses and demonstrate the utility of 3D-TrIm for dissecting dynamic virus-cell interactions at high spatiotemporal resolution.

Nanoparticle diffusion in 3D porous structures is critical to understanding natural and synthetic systems but remains underexplored due to limitations in traditional microscopy methods. Here, 3D Single‐Molecule Active‐feedback Real‐time Tracking (3D‐SMART) microscopy is used to resolve nanoparticle dynamics in agarose gels with unprecedented spatiotemporal resolution. This highlights “hopping diffusion”, where particles intermittently escape confinement pockets, providing insights into hydrogel microstructure. Long, highly sampled trajectories enable the extraction of kinetic parameters, confinement sizes, and thermodynamic barriers. This study demonstrates 3D‐SMART's ability to probe particle‐environment interactions at super‐resolution (≈10 nm in XY and ≈30 nm in Z) in 3D, offering new perspectives on nanoparticle diffusion and the structural dynamics of porous materials, with implications for drug delivery, material science, and biological systems.

Nanoparticle diffusion in 3D porous structures is critical to understanding natural and synthetic systems but remains underexplored due to limitations in traditional microscopy methods. Here, we use 3D Single-Molecule Active-feedback Real-time Tracking (3D-SMART) microscopy to resolve nanoparticle dynamics in agarose gels with unprecedented spatiotemporal resolution. We highlight ‘hopping diffusion’, where particles intermittently escape confinement pockets, providing insights into hydrogel microstructure. Long, highly sampled trajectories enable extraction of kinetic parameters, confinement sizes, and thermodynamic barriers. This study demonstrates 3D-SMART’s ability to probe particle-environment interactions at super-resolution (∼10 nm in XY and ∼30 nm in Z) in 3D, offering new perspectives on nanoparticle diffusion and the structural dynamics of porous materials, with implications for drug delivery, material science, and biological systems. ToC figure:

A viral infection process covers a large range of spatiotemporal scales. Tracking the viral infection process with fluorescent labels over long durations while maintaining a fast sampling rate requires bright and highly photostable labels. StayGold is a recently identified green fluorescent protein that has a greater photostability and higher signal intensity under identical illumination conditions compared to existing fluorescence protein variants. Here, StayGold protein fusions were used to generate virus-like particles (StayGold-VLPs) to achieve hour-long 3D single-virus tracking (SVT) with 1000 localizations per second (kHz sampling rate) in live cells. The expanded photon budget from StayGold protein fusions prolonged the tracking duration, facilitating a comprehensive study of viral trafficking dynamics with high temporal resolution over long time scales. The development of StayGold-VLPs presents a simple and general VLP labeling strategy for better performance in SVT, enabling exponentially more information to be collected from single trajectories and allowing for the future possibility of observing the entire life cycle of a single virus.

The viral infection process covers a large range of spatiotemporal scales. Tracking the viral infection process with fluorescent labels over long durations while maintaining a fast sampling rate requires bright and highly photostable labels. StayGold is a recently identified green fluorescent protein that has a greater photostability and higher signal intensity under identical illumination conditions as compared to existing fluorescence protein variants. Here, StayGold protein fusions were used to generate virus-like particles (StayGold-VLPs) to achieve hour-long 3D single-virus tracking (SVT) with one thousand localizations per second (kHz sampling rate) in live cells. The expanded photon budget from StayGold protein fusions prolonged the tracking duration, facilitating a comprehensive study of viral trafficking dynamics with high temporal resolution over long timescales. The development of StayGold-VLPs presents a simple and general VLP labeling strategy for better performance in SVT, enabling exponentially more information to be collected from single trajectories and allowing for the future possibility of observing the whole life cycle of a single virus. Abstract Figure:

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.

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-particle 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. 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 cellular protrusions. Finally, we demonstrate how this new method can move single-particle 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. One-Sentence Summary: Active-feedback 3D single-particle tracking enables an unprecedented look at the early stages of virus-cell interactions.

Skeletal reorganization is a type of intriguing processes because of their interesting mechanism, high atom-economy and synthetic versatility. Herein, we describe an unusual, divergent skeletal reorganization of N-sulfonyl ynamides. Upon treatment with lithium diisopropylamine (LDA), N-sulfonyl ynamides undergo a skeletal reorganization to deliver thiete sulfones, while the additional use of 1,3-dimethyl-tetrahydropyrimidin-2(1H)-one (DMPU) shifts the process to furnish propargyl sulfonamides. This skeletal reorganization divergence features broad substrate scope and scalability. Mechanistically, experimental and computational studies reveal that these processes may initiate from a lithiation/4-exo-dig cyclization cascade, and the following ligand-dependent 1,3-sulfonyl migration or β-elimination would control the chemodivergence. This protocol additionally provides a facile access to a variety of privileged molecules from easily accessible ynamides.

Indoles are ubiquitous structures that are found in natural products and biologically active molecules. The synthesis of indoles and indole‐involved synthetic methodologies in organic chemistry have been receiving considerable attention. Indole‐ N ‐carboxylic acids and derived indole‐ N ‐carboxamides are intriguing compounds, which have been widely used in organic synthesis, especially in multicomponent reactions and C−H functionalization of indoles. This Minireview summarizes the advances of reactions involving indole‐ N ‐carboxylic acids and indole‐ N ‐carboxamides in organic chemistry, and discusses the synthetic potential and perspective of this field.