Converting a conventional fluorescence microscope into a high-performance platform for sub-10-nm single-molecule localization microscopy

State-of-the-art super-resolution fluorescence microscopy enables visualization of molecular organization in intact cells with sub-10-nm resolution. Among these techniques, DNA-PAINT stands out for its conceptual simplicity, sub-5-nm lateral localization precision over large (100 × 100 μm²) fields of view, and the ability to multiplex up to 30 targets. However, achieving optimal performance typically requires a stable, focus-stabilized total internal reflection fluorescence (TIRF) system, which is often complex and costly for most life sciences laboratories. Here, we present a cost-effective and straightforward approach to convert a standard commercial epifluorescence microscope—commonly available in light microscopy facilities—into a high-performance TIRF platform. Our design features a novel focus-lock mechanism optimized for lateral sub-10-nm DNA-PAINT and (d)STORM super-resolution imaging. Using 2D and 3D DNA origami nanostructures and multicolor neuronal samples, we demonstrate image quality and localization precision comparable to state-of-the-art dedicated systems. This accessible method extends the reach and impact of high-end single-molecule localization microscopy, enabling nanoscale imaging capabilities in a wider range of laboratories. Joaquin Garat and colleagues present a cost-effective strategy to transform standard epifluorescence microscopes into focus-stabilized TIRF platforms for 2D and 3D single-molecule localization microscopy. The system delivers sub-10-nm DNA-PAINT and (d)STORM performance on par with state-of-the-art instruments, broadening access to high-quality nanoscale imaging.