The Nucluear Pore Complex as Intrinsic Reporter for Isotropic Expansion Microscopy

Expansion microscopy (ExM) is a super-resolution imaging method that does not require any special optical microscope(1). The key point resides on the possibility of uniformly chemically expanding a sample, thus increasing the relative distances among objects of interest as fluorescent molecules labeling specific components. ExM is highly invasive; it involves gelation and digestion steps that could introduce artifacts and heterogeneities in the relative spatial distribution of complex proteins in the cells. The possibility to combine STED(2) and ExM (ExSTED)(3) allows not only an unprecedented resolution but also a higher sensitivity to discover possible pitfalls. 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)(4). In particular, we show that Nup153, a filamentous subunit localized in the nuclear pore basket(5), is an excellent reporter to address the isotropy of the expansion process quantitatively. The quantitative analysis carried out on NPCs, at different spatial scales, allows concluding that expansion microscopy can be used at the nanoscale with consistent accuracy in the range of 20 nm. It is an excellent method for structural studies of macromolecular complexe