Enigma at the nanoscale: can the NPC act as an intrinsic reporter for isotropic expansion microscopy?

Expansion microscopy is a super-resolution method that allows expanding uniformly biological samples, by increasing the relative distances among fluorescent molecules labeling specific components. The main \"enigma\" regarding this approach is given by the isotropic behavior at the nanoscale. The present study aims to determine the robustness of such a technique, quantifying the expansion parameters i.e. scale factor, isotropy, uniformity. Our focus is on the nuclear pore complex (NPC), as well-known nanoscale component endowed of a preserved and symmetrical structure localized on the nuclear envelope. Here, we show that Nup153 is a good reporter to quantitatively address the isotropy of the expansion process. The quantitative analysis carried out on NPCs, at different spatial scales, allows concluding that expansion microscopy can be used at the nanoscale with a uniform accuracy in the range of 20 nm. In addition, it is an excellent method for structural studies of macromolecular complexes.