<i>In Situ</i> Lighting-Up Near-InfraredMetabolic Probes for Super-Resolution Imaging of Glycogen

High-fidelity tracking of glycogen dynamics in living biosystems is critical for exploring the biological role of glycogen metabolism in diseases. However, <i>in situ</i> information on the glycogen state mainly relies on a glucose uptake fluorescence probe 2-NBDG, which has proven to be extremely limited owing to the “always-on” fluorescence, short emission wavelength, and low signal-to-noise (S/N) ratio. Herein, we for the first time demonstrate a metabolic-activated off–on probe for glycogen through covalently attaching a molecular rotor with hydrophilic glucose at the C-2 position to guarantee good miscibility with a complete fluorescence-off state before metabolic incorporation into glycogen particles. The probe Glycogen-Red achieves negligible background fluorescence (1/30 times than 2-NBDG) and an ultrahigh S/N ratio (61-fold than 2-NBDG) of lighting-up near-infrared (NIR) fluorescence in glycogen biosynthesis. Notably, our probe has the unique characteristic of bypassing the washing steps, offering a powerful toolbox for real-time tracking of glycogen biosynthesis and super-resolution mapping of glycogen structures in living cells.