High-specificity real-time tracking of single virus particles in the eye

Viruses are versatile platforms for gene therapy, vaccine delivery, and neuromodulation, but fully harnessing their potential in the nervous system requires precise visualization of single-particle dynamics during entry, trafficking, and genome release. This remains challenging because viral vectors are nanoscale, optically transparent, and typically labeled with fluorophores that generate high background from unbound probes. We present a carbon quantum dot (QD) virus co-assembly strategy that labels viral particles during native biogenesis, producing bright, photostable reporters while preserving viral integrity and tropism. Combining density gradient ultracentrifugation with size-selective filtration, we obtain highly purified, traceable viral preparations that are effectively free of unbound QDs, enabling high signal-to-background imaging. Using adeno-associated virus (AAV) as a nonenveloped vector and lentivirus as an enveloped vector, we achieve single-particle tracking of viral entry and intracellular trafficking in live cells, as well as in vivo visualization of gene delivery to ocular tissues as a model neural target. This carbon QD-enabled platform supports high-spatiotemporal-resolution viral imaging with minimal background and provides a generalizable approach for optical tracking and optimization of viral gene delivery vectors for neural applications.