A Novel RNA-Light Probe for Structured-Illumination Microscopy Super-Resolution Imaging of Nucleolar Ultrastructure and Analysis of Nucleolar Dynamics

Real-time visualization of nucleolar dynamics is essential for elucidating its physiological functions and understanding the mechanisms underlying nucleolus-related stress and diseases. However, this task is technically challenging: super-resolution imaging is required to resolve the organelle’s intricate architecture and rapidly changing morphology. Two major bottlenecks remain─the scarcity of probes compatible with such nanoscopy and the exclusive focus on proteins, which leaves the complete nucleolar morphology invisible. Here, we report a novel quinoline derivative probe (QVT) that can rapidly “light up” cellular RNA and exhibits superior performance compared to the commercial dye SYTO RNA Select, including red emission, greater RNA selectivity and binding affinity, and enhanced photostability. These optical advantages facilitate super-resolution mapping of nucleolar ultrastructure using structured-illumination microscopy (SIM), particularly in clearly delineating RNA-rich subcompartments such as the dense fibrillar component and fibrillar centers. More importantly, QVT enables SIM to monitor nucleolar RNA dynamics in living cells during ferroptosis in real time and to investigate the previously elusive dynamics of nucleolus and nucleolar RNA in fatty liver disease. This study not only establishes a versatile chemical scaffold for next-generation RNA-targeted probes but also opens new avenues for mechanistic exploration of nucleolar biology in both health and disease.