Hour-Long, Kilohertz Sampling Rate Three-Dimensional Single-Virus Tracking in Live Cells Enabled by StayGold Fluorescent Protein Fusions

Abstract

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.

Single-Molecule Tracking in Live Cell without Immobilization or without Hydrodynamic Flow by Simulations: Thermodynamic Jitter

“…For many of these approaches, green fluorescent protein (GFP) and its spectral relatives are particularly well suited due to their high fluorescence yield and photobleaching properties in the intracellular environment [3]. And they can be fused to proteins in vivo [4,5]. As a result of these technological and biotechnological developments, single-molecule studies in liquids and live cells have emerged.…”