Subtle Structural Translation Magically Modulates the Super-Resolution Imaging of Self-Blinking Rhodamines

The evolution of super-resolution imaging techniques is benefited from the ongoing competition for optimal rhodamine fluorophores. Yet, it seems blind to construct the desired rhodamine molecule matching the imaging need without the knowledge on imaging impact of even the minimum structural translation. Herein, we have designed a pair of self-blinking sulforhodamines (STMR and SRhB) with the bare distinction of methyl or ethyl substituents and engineered them with Halo protein ligands. Although the two possess similar spectral properties (λ_ab, λ_fl, ϕ, etc.), they demonstrated unique single-molecule characteristics preferring to individual imaging applications. Experimentally, STMR with high emissive rates was qualified for imaging structures with rapid dynamics (endoplasmic reticulum, and mitochondria), and SRhB with prolonged on-times and photostability was suited for relatively "static" nuclei and microtubules. Using this new knowledge, the mitochondrial morphology during apoptosis and ferroptosis was first super-resolved by STMR. Our study highlights the significance of even the smallest structural modification to the modulation of super-resolution imaging performance and would provide insights for future fluorophore design.