Hour-long, kilohertz sampling rate 3D single-virus tracking in live cells enabled by StayGold fluorescent protein fusions.

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.