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Single‐cell imaging has unique advantages of maintaining the in situ physiological state, morphology, and microenvironment, becoming a powerful tool to unravel the nature of intracellular nucleic acids. The analysis of nucleic acids unprecedentedly demands the sub‐molecule details at segment or subunit, secondary structure and monomer levels, instead of just probing the sequence and the abundance of nucleic acids. Detection of nucleic acids at the sub‐molecule level requires higher specificity and higher sensitivity, which becomes a new challenge in nucleic acid analysis. Herein, we summarize the recent progress in the design and the application of single‐cell nucleic acid imaging methods at the sub‐molecule level, including the visualization of RNA splicing variants, RNA G‐quadruplexes in an individual gene, single nucleotide variation of mitochondrial DNA, and RNA m6A methylation. Remarkably, we highlight the key strategy, "Module Assembly", for high‐performance molecular recognition and demonstrate the required improvements in future research. [ABSTRACT FROM AUTHOR]

Imaging agents, molecular recognition, nucleic acids, single-cell imaging, sub-molecule level Frontispiece: Module Assembly Strategy for Single-Cell Nucleic Acid Imaging at the Sub-Molecule Level Here, the key strategy, "Module Assembly", for single-cell imaging of nucleic acids at the sub-molecule level is reviewed. [Extracted from the article]