Super-resolution imaging of the nuclear pore Free

Advances in electron microscopy (EM) have made it possible to elucidate the ultrastructure of organelles and the architecture of large complexes, including determination of the 3D structure of the nuclear pore complex (NPC), a large assembly of 30 nuclear proteins, with a resolution of only a few nanometres. However, owing to typical EM sample preparation methods, only a subset of target molecules is likely to be detected. To overcome this, EM can be used in combination with localisation microscopy, which allows accurate determination of molecular positions in the context of an ultrastructure provided by EM. To investigate possible heterogeneity of the NPC, Markus Sauer and colleagues (p. 4351) now use correlative scanning EM (SEM) combined with direct stochastic optical reconstruction microscopy (dSTORM) to image NPC components in isolated Xenopus laevis oocytes. The authors here make use of the periodic molecular structure of the NPC in order to locate proteins by using dSTORM and SEM, before superimposing the obtained images with high precision. With this approach, they are able to show that most NPCs contain eight gp210 homodimers, resulting in an eightfold symmetrical structure. Interestingly, a small subset of NPCs shows a ninefold symmetry, suggesting that there is some flexibility in NPC assembly. Taken together, this work clearly demonstrates the potential of combining EM with super-imaging approaches to unravel hitherto inaccessible molecular details, and such an approach is also likely to be applicable to other cellular structures.