High Specific Real-Time Tracking of Single Virus Particles.

Viruses are powerful biological platforms with broad applications in gene therapy, vaccine delivery, neuroscience, and nanomedicine. Unlocking their full potential requires a precise understanding of their dynamic behaviors, such as cellular entry, intracellular trafficking, and genome release, at the single-particle level. However, real-time tracking remains challenging due to the nanoscale size and optical transparency of viral particles, as well as limitations in current fluorescent labeling strategies. Conventional approaches using organic dyes or quantum dots (QDs) are often hindered by nonspecific background signals arising from unbound fluorophores. Here, we introduce a carbon quantum dot-virus association strategy that labels viral particles during their native assembly and packaging, thereby avoiding the limitations of postsynthetic modification. Through the integration of density gradient ultracentrifugation and size-selective filtration, we obtain highly purified, traceable viral particles devoid of detectable free QDs. Using adeno-associated virus (AAV) as nonenveloped virus models and lentivirus as enveloped ones, we demonstrate real-time tracking of viral entry, intracellular dynamics, and in vivo gene delivery to ocular tissues. This carbon QD-enabled platform presents single-particle spatiotemporal resolution and signal clarity, supporting high-fidelity viral imaging and next-generation viral therapies.