Modulating Fluorescence of Rhodamine through Molecular Rotor for Wash-free Living-cell Nanoscopy

Super-resolution imaging techniques that overcome the diffraction limit of light have gained wide popularity for visual-izing cellular structures with nanometric resolution. Although fluorogenic spirocyclization-structural rhodamine dyes remain the premier type of small-molecule fluorophore for super-resolution imaging owing to excellent photostability, high brightness and variable excitation from visible to near-infrared light. Unfortunately, they suffer from poor per-formance due to their propensity to adopt a lipophilic, nonfluorescent form. In this work, we proposed a molecular ro-tor based modular strategy to develop water-soluble fluorogenic probes (MG-Rho) by directly connecting spirocycliza-tion-free rhodamine dyes and malachite green (MG) through electronically conjugated bond. The underlying mecha-nism is investigated through photoluminescence spectra, time-resolved absorption spectra, as well as theoretical calcu-lations etc., which indicate that the free rotation of MG serves as a driving force to induce the excited-state non-radiative energy consumption. This work offers a versatile design strategy for developing wash-free super-resolution probes and introduces a mechanical mechanism for modulating excited-state photophysical processes at the molecular level.