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.