Kinetic design of reversible probe exchange enables continuous single-molecule tracking beyond the photobleaching limit

Abstract Single-molecule fluorescence imaging enables direct tracking of molecular dynamics. Prolonged observation, however, is limited by photobleaching, restricting access to rare events and slow transitions. Exchangeable fluorogenic labeling has been developed to overcome this limitation, yet continuous single-molecule tracking has not been achieved. We define two quantitative kinetic conditions for continuous tracking: pre-bleach dissociation and within-frame rebinding. Mapping reported systems onto a kinetic landscape delineates the regime required for continuous tracking. To reach this regime, we turned to odorant-binding proteins, whose biological role favors rapid ligand capture and release. We paired this scaffold with high-contrast fluorogenic probes to maximize rebinding capacity while preserving rapid dissociation. The resulting system, EverGreen, sustains uninterrupted single-molecule observation for over 24 hours and establishes a general kinetic design framework beyond the photobleaching limit.