Combining pMINFLUX, Graphene Energy Transfer and DNA-PAINT for Nanometer Precise 3D Super-Resolution Microscopy

3D super-resolution microscopy with nanometric resolution is a key to fully complement ultrastructural techniques with fluorescence imaging. Here, we achieve 3D super-resolution by combining the 2D localization of pMINFLUX with the axial information of graphene energy transfer (GET) and the single-molecule switching by DNA-PAINT. We demonstrate <2 nm localization precision in all 3 dimension with axial precision reaching below 0.3 nm. In 3D DNA-PAINT measurements, structural features, i.e., individual docking strands at distances of 3 nm, are directly resolved on DNA origami structures. pMINFLUX and GET represent a particular synergetic combination for super-resolution imaging near the surface such as for cell adhesion and membrane complexes as the information of each photon is used for both 2D and axial localization information. Furthermore, we introduce local PAINT (L-PAINT), in which DNA-PAINT imager strands are equipped with an additional binding sequence for local upconcentration improving signal-to-background ratio and imaging speed of local clusters. L-PAINT is demonstrated by imaging a triangular structure with 6 nm side lengths within seconds.