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 with moderate success. Here, we present optimized methods for phalloidin conjugate grafting that have a high efficiency for both cellular and tissue samples. Our optimized strategy improves anchoring and signal retention by ∼10 times. We demonstrate the potential of optimized trifunctional linkers (TRITON) for actin imaging in combination with immunolabeling using different ExM protocols. 10X ExM of actin labeled with optimized 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 in which the concentration of target-bound monomers is insufficient for high-quality ExM.

Expanding boundaries – a cell biologist's guide to expansion microscopy

“…Recently, glycidyl methacrylate has been proposed for the effective anchoring of protein and RNA simultaneously ( Cui et al, 2023 ). Furthermore, to directly crosslink (graft) only target molecules to the sample and label them at the same time, multifunctional dyes have been developed ( Thielhorn et al, 2023 ; Wen et al, 2020 , 2023b ).…”

“…Consequently, the incorporation of monomers on the surface of target molecules into the polymerizing gel (surface grafting) is relatively unlikely, as polymerization occurs through diffusion-mediated collision. To effectively link all material of the sample into the polymerizing gel, a high concentration of anchoring agent must be used in sample preparation, or several anchors must be attached to a target molecule ( Thielhorn et al, 2023 ; Wen et al, 2023b ).…”

Bio-orthogonal Glycan Imaging of Culture Cells and Whole AnimalC. eleganswith Expansion Microscopy

“…To enhance fluorescence and probe retention, multifunctional chemical linkers that directly graft fluorophores to ExM gels have recently been demonstrated for coupling various biomolecules including protein tags 31,32 , lipids 33,34 , and actin 35 . These approaches, parallelly termed trivalent anchoring (TRITON) 33 and label-retention expansion microscopy (LR-ExM) 31 , are attractive candidates for probing macromolecules in multicellular systems due to the small size of the reagents.…”