Super-resolution microscopy heads towards 3D dynamics

Abstract Resolving fine details of subcellular structures is key to understanding the organization and function of cellular networks. Recent advances in far-field fluorescence microscopy provide the necessary tools to analyze these structures with resolutions well below the classical diffraction limit in all three dimensions. Technical improvements go hand-in-hand with new versions of switchable fluorophores that allow nonlinear optical effects to be more efficiently used to push the resolution limit down further. High contrast combined with the wide spectrum of available colors currently endow these fluorescence-based super-resolution techniques with the power to study the complexity of subcellular organelles and the relation of their constituting components down to the molecular level and under physiological conditions. In this way, they give us a far better understanding of the assembly of macromolecular complexes and their functions within a cell than has been possible before employing conventional imaging methods. In this review, we give an overview of the technical state-of-the art of these technologies, their fundamental and technical trade-offs, and provide typical application examples in this exciting field.