De Novo Designed Self-Assembling Rhodamine Probe for Real-Time, Long-Term and Quantitative Live-Cell Nanoscopy

Super-resolution imaging provides a powerful approach to image dynamic biomolecule events at nanoscale resolution. An ingenious method involving tuning intramolecular spirocyclization in rhodamine offers an appealing strategy to design cell-permeable fluorogenic probes for super-resolution imaging. Nevertheless, precise control of rhodamine spirocyclization presents a significant challenge. Through detailed study of the structure-activity relationship, we identified that multiple key factors control rhodamime spirocyclization. The findings provide opportunities to create fluorogenic probes with tailored properties. On the basis of our findings, we constructed self-assembling rhodamine probes for no-wash live-cell confocal and super-resolution imaging. The designed self-assembling probe <b>Rho-2CF3</b> specifically labeled its target proteins and displayed high ring-opening ability, fast labeling kinetics (<1 min), and large turn-on fold (>80 folds), which is very difficult to be realized by the existing methods. Using the probe, we achieved high-contrast super-resolution imaging of nuclei and mitochondria with a spatial resolution of up to 42 nm. The probe also showed excellent photostability and proved ideal for real-time and long-term tracking of mitochondrial fission and fusion events with high spatiotemporal resolution. Furthermore, <b>Rho-2CF3</b> could resolve the ultrastructure of mitochondrial cristae and quantify their morphological changes under drug treatment at nanoscale. Our strategy thus demonstrates its usefulness in designing self-assembling probes for super-resolution imaging.