A highly homogeneous expansion microscopy polymer composed of tetrahedron-like monomers

Expansion microscopy (ExM) physically magnifies biological specimens to enable nanoscale-resolution imaging on conventional microscopes. Current ExM methods permeate biological specimens with free radical-polymerized polyacrylate hydrogels, whose network structure limits the microscopy resolution enabled by ExM. Here we report that ExM is possible using hydrogels with more homogeneous network structure, assembled via non-radical terminal linking of monomers of tetrahedral shape. As with earlier forms of ExM, such \"tetra-gel\"-embedded specimens can be iteratively expanded for greater physical magnification. Iterative tetra-gel expansion of herpes simplex virus type 1 (HSV-1) virions by ~10x in linear dimension results in a viral envelope deviation from sphericity of 9.2 nm, rather than the 14.3 nm enabled by free radical-polymerized hydrogels used in earlier versions of ExM. Thus, tetra-gel polymer chemistry may support new forms of ExM imaging that introduce fewer spatial errors than earlier versions, and raise the question of whether single biomolecule precision may be achievable.