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

Abstract

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 (logP) over a wide range and confirmed that SNR can be sharply improved, owing to the promoted nano-aggregation (K_A/M) with high logP. 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_vβ₃ 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.

Keywords

fluorescence imaging / nano-aggregates / signal-to-noise ratio / substituted rhodamine / super-resolution microscopy

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