Advancement of Peptide‐PAINT: Applications and Current Challenges in Super‐Resolution Imaging

Super‐resolution fluorescence microscopy has revolutionized the ability to probe biomolecular organization at the nanoscale in both fixed and living cells. Among various single‐molecule localization microscopy approaches, Point Accumulation for Imaging in Nanoscale Topography (PAINT) offers a conceptually distinct framework by leveraging transient probe–target interactions rather than fluorophore photo‐physics. Building on implementations such as DNA‐PAINT, Peptide‐PAINT has emerged as a versatile and accessible strategy for imaging protein organization with high specificity. This review provides a comprehensive overview of Peptide‐PAINT, covering its fundamental principles and recent methodological advances, including exchangeable, fluorogenic, and endogenous labeling strategies. We highlight its capability for multiplexed imaging and discuss emerging applications in live‐cell systems. Furthermore, we critically evaluate current limitations, including probe kinetics, orthogonality of peptide pairs, specificity, and intracellular delivery. Finally, we outline future directions for engineering robust, easy‐to‐implement Peptide‐PAINT platforms for quantitative and high‐throughput super‐resolution imaging.