The last two decades have seen the advent of a variety of super-resolution imaging techniques that provide spatial resolution well below the diffraction limit, approaching virtually molecular detail. Their widespread application is currently transforming biological research enabling new and exciting views on the structural organisation of cells and the dynamics of biomolecular assemblies on different timescales. These revolutionary developments come with novel requirements for fluorescent probes, labelling techniques, and data interpretation strategies. Researchers from across the scientific spectrum are involved in designing improved tools and solving previously intractable problems using super-resolution techniques.