Visualization of F-Actin Through Expansion Microscopy (ExM) with Trifunctional Linker-Conjugated Phalloidin.

Expansion microscopy (ExM) is an imaging technique that enables super-resolution imaging of biological specimens using conventional confocal microscopy. This process entails the isotropic physical expansion of a (biomolecular) sample that has been cross-linked to a swellable polymer. The grafting of biomolecules (and the subsequent fluorescent readout) is accomplished by introducing an acryloyl group to the amine groups of lysine residues within the proteins, enabling subsequent imaging. However, visualizing actin filaments with high spatial resolution using ExM remains challenging. Herein, we report the construction of a phalloidin conjugate containing actin stains and their application in ExM. This protocol highlights the efficacy of trifunctional linker (TRITON/Actin-ExM) for F-actin imaging, demonstrating that TRITON-labeled actin allows for efficient anchoring and signal retention, enabling robust visualization of actin filaments in expansion microscopy. Key features • Engineered linker (TRITON) design ensures efficient fluorophore attachment, resulting in bright, stable signals during imaging. • Performed pre-expansion and antibody-free labeling. • Detailed and specific visualization of actin filaments in ExM experiments (4-fold expansion).