Super-resolution microscopy has transformed our ability to probe cellular organization at the molecular scale. Among these approaches, DNA-PAINT (Points Accumulation for Imaging in Nanoscale Topography) provides spatial resolution down to the single-protein level, supports straightforward multiplexing, and enables molecular counting. DNA-PAINT relies on fluorescently labeled oligonucleotides that diffuse and stochastically bind to their complementary “docking strands” attached to target molecules. This equilibrium of binding and unbinding is both well characterized and highly programmable in sequence space, making DNA-PAINT a powerful tool for advanced single-molecule microscopy applications.

In the first part of my talk, I will introduce the core principles of DNA-PAINT and highlight recent developments in the field, including our own contributions. These include the design of a tailored microscope, ultraprecise kinetic measurements that enable robust multiplexing and counting, and new single-particle-tracking implementations.

In the second part of my talk, I will outline my current biological focus: refining and applying the quantitative DNA-PAINT toolbox to uncover how nuclear organization shapes genome function. By visualizing protein–RNA–DNA interactions with nanometer precision, we aim to understand the molecular principles that govern nuclear compartmentalization, many of which are linked to liquid-liquid phase separation. This pursuit has led us to the interface with electron microscopy, where integrating molecular mapping with ultrastructural context opens new opportunities for in situ structural biology.