Visualizing Nucleolar Abnormality in Human Cancer Tissues With a Fluorescent RNA Probe for Intraoperative Pathological Diagnosis

Accurately distinguishing malignant from benign tumors within 30 min is an essential yet challenging task for intraoperative pathological diagnosis. However, due to the lack of an efficient fluorophore capable of rapid and precise imaging of nucleoli in human tissues, the clinical diagnostic value of nucleoli remains underexplored, although abnormal nucleolar morphology in cancer cells represents a particularly prominent feature of malignant tumors. Given that nucleoli contain evolutionarily conserved and abundant ribosomal RNA (rRNA), their morphology in various cell types, including normal, benign, and cancerous cells, should be able to be imaged precisely and rapidly by a fluorescent RNA probe with high RNA affinity and tissue permeability. Herein, we developed a fluorescent probe, SDU‐CH1, by integrating a pyridinium cation bearing a terminal ‐NH 3 + group with a carbazole moiety. Fluorescence spectra and molecular docking calculations revealed that SDU‐CH1 exhibited ultra‐high affinity for conserved and abundant 18S rRNA through electrostatic interactions, π‐π interaction, and hydrogen bonds. Benefiting from exceptional RNA affinity and tissue permeability, SDU‐CH1 enabled rapid staining and high‐fidelity imaging of nucleoli and cytoplasm in diverse cells within frozen sections of human breast tumor, particularly in ductal epithelial cells where most breast cancer originates. Furthermore, fluorescence imaging of these frozen sections not only allowed clear visualization of nucleolar morphology and abnormality, but also facilitated quantitative assessment of nucleolar area and nucleolar/nuclear area ratio. Importantly, these nucleolar abnormalities and quantitative indicators could be effectively employed to identify malignant tumors, contributing a new method for intraoperative pathological diagnosis.