Beyond 4×: pathways to higher expansion factors in expansion microscopy

Expansion microscopy (ExM) enables nanoscale imaging with conventional light microscopes by physically magnifying biological specimens. While classical ExM achieves ∼4× linear expansion, advances in polymer chemistry and workflow design now routinely push expansion factors beyond 4×, thereby improving resolution while retaining compatibility with diverse labelling strategies. Here, we review current one-step and multi-step (iterative) high-expansion approaches, outlining how modifications to monomer composition yield highly swellable gels, and how iterative embedding can bypass the mechanical limits of any single polymer network. Finally, we discuss practical considerations and highlight how emerging strategies could maximise swelling capacity and simplify workflows. Together, these developments move the field toward scalable, reproducible, and broadly accessible nanoscale imaging.