A bright and regenerative array tag with fluorogenic ligands for long-term live-cell nanoscopy and single-molecule tracking

Live-cell fluorescence nanoscopy and single-molecule tracking offer profound insights into cellular biology, but their application is limited by fluorescent tags with low brightness, large size, and irreversible photobleaching. Here, we present ReTagX, a bright, regenerative, compact array tag system enabling rapid recovery of photobleached signals through reversible, fluorogenic labeling. Newly developed ReTag1 (~18 kDa) with highly fluorogenic MaP655-TMP achieves a signal-to-noise ratio up to 204-fold and allows fluorescence recovery within 1 minute in wash-free live-cell imaging. This tag extends STED imaging of mitochondrial TOM20 from fewer than 5 to over 300 frames, enabling long-term visualization of mitochondrial dynamics at 80 nm resolution for over 100 minutes. Multimeric ReTag1 arrays, such as ReTag8 (~156 kDa), increase brightness 5.5-fold and extend TNFR2 single-molecule tracking ~2.9-fold, preserving >80% of trajectories over 5 minutes. ReTagX provides a robust platform for high-resolution, long-term molecular visualization of cellular dynamics in living cells.