Expansion microscopy at the nanoscale: The nuclear pore complex as a fiducial landmark

Expansion microscopy (ExM) is a magnification method that allows achieving super-resolved images using a conventional light microscope. In ExM, biomolecules, fluorescent proteins, and dyes are functionalized with specific handles to link a dense polyelectrolyte hydrogel, which can achieve an isotropic expansion of 4.5-fold in water. The use of ExM coupled with STED nanoscopy allows examining macromolecular machinery in life science, like the nuclear pore complex (NPC). In particular, in this chapter, we show a general protocol for labeling one of its subunit, i.e. the Nup153. Such method shows the nanoscale isotropy of the expansion process and enables precise measurement of the expansion factor. Finally, we used ExM for the visualization of a peculiar nuclear invagination in normal and aged cells.