Advances in super-resolution microscopy (SRM) have made it possible to visualise cellular processes with exquisite detail and provided important insights into the nanoscale organisation of numerous proteins. Nevertheless, SRM approaches to analyse nuclear signalling pathways have been limited owing to the imaging depth required and low throughput of the method. Expansion microscopy (ExM) has the potential to overcome these limitations but has not yet been widely applied to the nucleus because of concerns over differential anisotropic expansion. Joanna Morris, Robert Neely and co-workers (Faulkner et al., 2022) now develop conditions for the isotropic expansion of the nucleus of human epithelial cells with minimal distortion; this involves the anchoring of nucleic acids into the expansion gel, yielding a resolution of 120–130 nm after an approximate four-fold expansion. To validate the method, they analyse the 3D organisation of thousands of DNA repair foci formed upon induction of DNA damage by following three key factors. This enables them to observe unprecedented details regarding substructure heterogeneity. Furthermore, changes in foci structure and composition in response to depletion of chromatin regulators are also detectable by ExM. Taken together, this work demonstrates that ExM allows for a quantitative description of the spatial organisation of multiple proteins within nanoscale nuclear structures, thus greatly expanding the nuclear imaging toolbox.
