Fluorogenic speed-optimized DNA-PAINT probes enable super-resolution imaging of whole cells

Super-resolution microscopy with DNA-PAINT enables molecular-scale, multiplexed, and quantitative imaging, but its throughput is limited by slow binding kinetics and elevated background at high probe concentrations. Recent speed-optimized and fluorogenic probes improve performance but impose strong constraints on sequence design, revealing a fundamental tradeoff between fast binding and efficient quenching. Here, we introduce a modular probe architecture that spatially decouples binding kinetics from fluorophore–quencher interactions by integrating speed-optimized sequence motifs with PEG spacers. Using DNA origami nanostructures, we demonstrate enhanced localization rates, signal-to-background ratios, and imaging efficiency compared to state-of-the-art probes. We validate our approach in cells, demonstrating its capability to image nuclear targets and enabling three-dimensional imaging of the endoplasmic reticulum using standard widefield illumination. Our work establishes a general framework for fast, multiplexed, and low-background super-resolution imaging.