Commonly applied super-resolution light microscopies have provided insight into subcellular processes at the nanoscale. However, imaging depth, speed, throughput and cost remain significant challenges, limiting the numbers of three-dimensional (3D) nanoscale processes that can be investigated and the number of laboratories able to undertake such analysis. Expansion microscopy (ExM) solves many of these limitations, but its application to imaging nuclear processes has been constrained by concerns of unequal nuclear expansion. Here, we demonstrate the conditions for isotropic expansion of the nucleus at a resolution equal to or better than 120-130 nm (pre-expansion). Using the DNA damage response proteins BRCA1, 53BP1 (also known as TP53BP1) and RAD51 as exemplars, we quantitatively describe the 3D nanoscale organisation of over 50,000 DNA damage response structures. We demonstrate the ability to assess chromatin-regulated events and show the simultaneous assessment of four elements. This study thus demonstrates how ExM can contribute to the investigation of nanoscale nuclear processes.
Keywords: 53BP1; BRCA1; DNA damage; Expansion microscopy; Nanoscale; RAD51.
© 2022. Published by The Company of Biologists Ltd.