The actin cytoskeleton has a central role in cell biology, supporting essential processes such as cell shape maintenance, migration and division, and intracellular transport. Its strong evolutionary conservation reflects both its fundamental cellular functions and the structural constraints that govern the organization of actin filaments. Visualizing actin architecture has therefore long been a priority; this has traditionally relied on antibodies or small-molecule probes such as phalloidin that selectively bind filamentous actin (F-actin). However, actin filaments (~7 nm in diameter) assemble into dense, heterogeneous networks that largely remain below the diffraction limit of conventional light microscopy, which makes super-resolution approaches essential for resolving their nanoscale organization.

To address this need, we have developed HAK-actin by fusing a haemagglutinin (HA) tag and lysine (K) residues to a probe for efficient actin labelling in U-ExM. The lysine residues effectively anchor the probe to the swellable hydrogel matrix, enabling post-expansion labelling of the HA tag that enhances signal retention and amplification. It performs robustly across both chemically fixed samples and cryogenically fixed samples, supporting U-ExM, cryo-ExM and iterative expansion (iU-ExM) workflows. Its labelling density is comparable to that of conventional anti-actin antibodies, while it offers improved compatibility across phylogenetically diverse eukaryotic systems, from mammalian cells and tissues to microbial models.