Expansion microscopy of neutrophil nuclear structure and extracellular traps

Neutrophils are key players of the immune system and possess an arsenal of effector functions, including the ability to form and expel neutrophil extracellular traps (NETs) in a process termed NETosis. During NETosis, the nuclear DNA/chromatin expands until it fills the whole cell and is released into the extracellular space. NETs are composed of DNA decorated with histones, proteins, or peptides, and NETosis is implicated in many diseases. Resolving the structure of the nucleus in great detail is essential to understand the underlying processes, but so far, superresolution methods have not been applied. Here, we developed an expansion-microscopy-based method and determined the spatial distribution of chromatin/DNA, histone H1, and nucleophosmin with an over fourfold improved resolution (<40-50 nm) and increased information content. It allowed us to identify the punctate localization of nucleophosmin in the nucleus and histone-rich domains in NETotic cells with a size of 54-66 nm. The technique could also be applied to components of the nuclear envelope (lamins B1 and B2) and myeloperoxidase, providing a complete picture of nuclear composition and structure. In conclusion, expansion microscopy enables superresolved imaging of the highly dynamic structure of nuclei in immune cells.