Super-resolution labelling with Action-PAINT

Recent advances in localisation-based super-resolution microscopy have enabled researchers to visualise single molecular features down to individual molecular components (~5 nm), but do not yet allow manipulation of single-molecule targets in a user-prescribed, context-dependent manner. Here we report an “Action-PAINT” strategy for super-resolution labelling upon visualisation on single molecules. This approach monitors and localises DNA binding events in real-time with DNA-PAINT, and upon visualisation of binding to a desired location, photo-crosslinks the DNA to affix the molecular label. We showed the efficiency of 3-cyanovinylcarbazole nucleoside (CNVK) photo-inducible crosslinking on single molecular targets and developed a software package for real-time super-resolution imaging and crosslinking control. We then benchmarked our super-resolution labelling method on synthetic DNA nanostructures and demonstrated targeted multi-point labelling on various complex patterns with 30 nm selectivity. Finally, we performed targeted in situ labelling on fixed microtubule samples with 40 nm target size and custom-controlled, sub-diffraction spacing.