For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.

Fluorescence light microscopy allows multicolor visualization of cellular components with high specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution. We applied three-dimensional structured illumination microscopy (3D-SIM) to circumvent this limit and to study the mammalian nucleus. By simultaneously imaging chromatin, nuclear lamina, and the nuclear pore complex (NPC), we observed several features that escape detection by conventional microscopy. We could resolve single NPCs that colocalized with channels in the lamin network and peripheral heterochromatin. We could differentially localize distinct NPC components and detect double-layered invaginations of the nuclear envelope in prophase as previously seen only by electron microscopy. Multicolor 3D-SIM opens new and facile possibilities to analyze subcellular structures beyond the diffraction limit of the emitted light.Light microscopy is a key technology in modern cell biology and, in combination with immunofluorescence, fluorescent protein fusions, or in situ hybridization, allows the specific localization of nearly all cellular components. A fundamental limitation of optical microscopy is its low resolution relative to the scale of subcellular structures. This limitation occurs because light traveling through a lens cannot be focused to a point but only to an Airy disk (1) with a diameter of about half the wavelength of the emitted light (2,3). Because the wavelengths of visible light range from 400 to 700 nm, objects closer than 200 to 350 nm apart cannot be resolved but appear merged into one.