AbstractFluorescent probes are valuable tools to visualize non‐catalytic proteins in live cells. Currently, the majority of imaging reagents for non‐catalytic proteins are based on “always‐on” fluorophores and the use of these reagents usually necessitate a wash step to remove unbounded fluorophores before microscope imaging. Herein, we report the use of arylamino‐substituted rhodamine as an activatable fluorophore for the imaging of non‐catalytic protein in live cells. We have shown the induction of an arylamino to structurally rigid rhodamine could significantly reduce the fluorescent emission in aqueous medium but the ligand‐directed binding of this molecule to protein receptor could effective restrict its intramolecular motion and thus lead to enhancement in fluorescence intensity at 590 nm over 30‐fold. With fluorescent probes based on this fluorophore, we could visualize integrin αvβ3 and azido‐functionalized glycans in living cells with high contrast in a wash‐free manner.

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