Subtle Structural Translation Remarkably Modulates the Super-Resolution Imaging of Self-blinking Rhodamines

Abstract

The evolution of super-resolution imaging techniques is benefited from the ongoing competition for optimal rhodamine fluorophores. Yet, it seems blinded to select the best one among different rhodamine derivatives for specific labeling and imaging, without the knowledge on imaging impact of even the minimum structural transform. Herein, we have designed a pair of self-blinking sulforhodamines (STMR, SRhB) with the bare distinction of methyl or ethyl substituents, and engineered them with Halo protein ligands. Although the two present similar spectral properties (λab, λfl, 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, mitochondria), and SRhB with prolonged on-times and photostability was suited for relatively "static" nuclei and microtubules. Utilized 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 insight for future fluorophore design.

Recruiting Rate Determines the Blinking Propensity of Rhodamine Fluorophores for Super-Resolution Imaging

“…Despite their different p K cycling (6.91, SRhB; 7.34, PySRh; 5.05, PiSRh), three rhodamines have adequate recruiting rates (>0.1 s –1 ) for localization imaging. The imaging results confirm that sulfonamide rhodamines are qualified for super-resolution imaging of various organelles in living cells (Figure ; see results of SRhB in another our study, which extends the topic to discuss the imaging disparities between different sulfonamide rhodamines).…”