Ether Rhodamines with Enhanced Hydrophilicity, Fluorogenicity, and Brightness for Super-Resolution Imaging

Rhodamine dyes are widely used fluorophores in super-resolution fluorescence imaging due to their exceptional optical properties and "aggregation-disaggregation" induced fluorogenic activation. However, their excessive lipophilicity often reduces brightness in aqueous environments and causes off-target staining, limiting their effectiveness in high-resolution imaging. To address these challenges, we introduce an ether-decorated N-terminal modification strategy for rhodamine and silicon-rhodamine (Si-rhodamine), replacing conventional <i>N</i>-alkyl groups. The ether chains enhance water solubility, decrease aggregate size, and improve fluorogenicity across a wide concentration range. Their flexible, hydrophilic structure forms a protective shield around the xanthene core, minimizing dye-water interactions and reducing fluorescence quenching. Additionally, the inductive effect of the ether chains decreases the electron-donating strength of the amino groups, suppressing quenching caused by twisted intramolecular charge transfer (TICT). These modifications collectively increase the quantum yields of <b>ER</b> and <b>ESiR</b> in water from 0.35 and 0.19 (for tetraethyl-substituted analogs) to 0.70 and 0.41, respectively. Probes derived from <b>ER</b> and <b>ESiR</b> exhibit outstanding fluorogenicity, enhanced signal-to-noise ratios, and improved resolution in complex aqueous environments, demonstrating superior performance in advanced super-resolution imaging techniques such as structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, and single molecule localization microscopy (SMLM). This work introduces a rational fluorophore design strategy that enhances aqueous brightness, minimizes nonspecific staining, and enables high-contrast, high-resolution imaging across multiple super-resolution modalities.