A General Strategy to Fine‐Tune Group 14 Rhodamines for Ultrahigh Signal‐to‐Noise Ratio Labeling In Vivo by Nano‐Aggregation

Ultrahigh signal‐to‐noise ratio (SNR) labeling enables precise visualization of biological structures in vivo. We boosted fluorogenicity in group‐14‐rhodamines by comprehensively manipulating their dynamics in physical (aggregate/monomer, K A/M ) and chemical (closed/open spirolactone, K C/O ) states. Fluorogenic rhodamines were designed by group 14 (C, Si, Ge) substituted bridging regions in xanthene with tuned dialkylation. We quantified the impact of alkylation with the hydrophobicity (log P ) over a wide range and confirmed that SNR can be sharply improved, owing to the promoted nano‐aggregation ( K A/M ) with high log P . Integrating K A/M with K C/O mechanisms, unparalleled fluorogenicity was observed in group‐14‐rhodamines: HaloTag probe with dipentylsilyl exhibits remarkable fluorogenicity (>2000) in vitro, enabling no‐wash and multicolor super‐resolution stimulated emission depletion imaging of high SNR (>300) in vivo. Overexpression of α v β 3 was sensitively tracked in vivo by RGDyK‐based fluorogenic SiR probe through tuned K A/M . Our proposed strategy has significantly promoted the fluorogenicity of group 14 rhodamines as a general mechanism.