Biocompatible sulfonium-based covalent probes for endogenous tubulin fluorescence nanoscopy in live and fixed cells

Fluorescent probes enable precise visualization of dynamic cellular processes, especially when combined with super-resolution imaging techniques that overcome the diffraction limit. However, traditional labeling strategies, including fluorescent protein fusions (e.g., GFP) or ligand-linked fluorophores, often perturb protein function or induce biological side effects. Here, we report a covalent fluorescent probe for endogenous tubulin, a key cytoskeletal protein governing cell division, motility, and intracellular transport. Using cabazitaxel as a tubulin targeting moiety and silicon-rhodamine as a cell permeable fluorophore, we designed and optimized probe, 6-SiR-o-C9-CTX, containing a biocompatible cleavable linker with a sulfonium center. It exhibits cell permeability, fluorogenic behavior, and efficient covalent labeling of tubulin across multiple human cell lines. Importantly, taxane targeting moiety can be removed post-labeling, preserving tubulin’s functions. This labeling strategy is compatible with STED nanoscopy in both live and fixed cells, enabling high-resolution, minimally invasive cytoskeletal imaging, and advancing the toolkit for studying dynamic cellular processes.