Hydrogel Chemistry in Expansion Microscopy: From Polyacrylamide to Next‐Generation Swellable Polymers

Expansion microscopy (ExM) is a transformative biological imaging approach that achieves nanoscale resolution on conventional diffraction-limited microscopes by physically expanding specimens embedded in swellable hydrogels. Since its introduction in 2015, the hydrogel chemistry underpinning ExM has evolved considerably, progressing from the original sodium polyacrylate/polyacrylamide (PAAm) formulations toward advanced polymer architectures that address persistent limitations in expansion factor, isotropy, and structural fidelity. This review comprehensively analyzes the hydrogel chemistry landscape in ExM, tracing the progression from classical polyacrylamide-based gels through modified acrylamide formulations and high-expansion dimethylacrylamide (DMAA) systems to next-generation click-chemistry-based tetra-gel platforms. We critically examine the polymerization mechanisms, monomer composition, crosslinker design, and anchoring strategies governing gel performance, and evaluate how these parameters influence expansion factor, mechanical integrity, and biomolecular spatial fidelity. Emerging approaches, including single-shot 20-fold expansion protocols and microwave-accelerated workflows, are discussed in the context of their underlying polymer chemistry. By synthesizing findings from the primary literature through 2025, this review aims to guide researchers in selecting and optimizing hydrogel systems for specific imaging applications and to identify promising directions for future polymer innovation in ExM.