Abstract
Fluorescent probes enable the visualization of dynamic cellular processes with high precision, particularly when coupled with super-resolution imaging techniques that surpass the diffraction limit. Traditional methods include fluorescent protein fusion (e.g., GFP) or organic fluorophores linked to ligands targeting the protein of interest. However, these approaches often introduce functional disruptions or ligand-associated biological effects. Herein, we address these challenges by developing covalent fluorescent probes for endogenous tubulin, a critical cytoskeletal protein involved in processes such as cell movement, division, and biomolecule trafficking. Using well-known tubulin binder cabazitaxel and cell permeable fluorophore silicon-rhodamine-as a basis, we introduce a novel biocompatible cleavable linker containing a sulfonium center. This allowed the construction of the optimized probe, 6-SiR-o-C 9 -CTX, demonstrating excellent cell permeability, fluorogenic properties, and the ability to covalently label tubulin across various human cell lines. Importantly, the targeting moiety could be washed out while preserving tubulin staining, ensuring minimal disruption of tubulin function. This labeling technique is compatible with STED nanoscopy in both live and fixed cells, offering a powerful high-resolution imaging tool.