Trifunctional linkers enable improved visualization of actin by expansion microscopy

ABSTRACT Expansion Microscopy (ExM) revolutionized the field of super-resolution microscopy by allowing for subdiffraction resolution fluorescence imaging on standard fluorescence microscopes. However, it has been found that it is hard to visualize actin filaments efficiently using ExM. To improve actin imaging, multifunctional molecules have been designed, however, with moderate success. Here, we present optimized methods for phalloidin conjugate grafting that have high efficiency for both cellular and tissue samples. Our optimized strategy improves anchoring and signal retention by ∼10 times. We demonstrate the potential of trifunctional linkers (TRITON) for actin imaging in combination with immunolabeling using different ExM protocols. 10x ExM of actin labeled with TRITON enabled us to visualize the periodicity of actin rings in cultured hippocampal neurons and brain slices by Airyscan confocal microscopy. Thus, TRITON linkers provide an efficient grafting method, especially in cases where the concentration of target-bound monomers is insufficient for high-quality ExM.