Photoclick Protein Anchoring for Expansion Microscopy

Expansion microscopy is a promising optical imaging technique with subdiffraction-limited resolution on conventional fluorescence microscopes, but it remains difficult to achieve efficient and controllable protein anchoring for rapid expansion imaging applications. Herein, we report a robust photoclick protein anchoring strategy-based expansion imaging technique, which allows minute-scale molecule anchoring and sample homogenization at the 1 h scale in cell and tissue samples. The photoclick protein anchoring strategy relies on the UV-light-induced cyclization reaction for anchor grafting onto the proteins, allowing subsequent protein anchoring to the hydrogel for expansion imaging. With this technique, we achieved the nanoscale feature imaging of microtubule networks and multiplexed imaging of structure reorganization of cellular cytoskeleton and cell nucleus with the presence of mechanosignaling disorder in vascular smooth muscle cells upon oxidized low-density lipoprotein (oxLDL) stimulation. We also validated the feasibility of our strategy for rapid protein anchoring and expansion imaging of hippocampal cell features in mouse brain slices. Moreover, we demonstrated photoclick protein anchoring in the region of interest (ROI) across the large-scale sample for selective sample retention and expansion imaging by using a mask-assisted exposure method. We believe that our method holds great promise as a robust candidate of super-resolution imaging techniques for versatile biological and clinical applications on standard fluorescence imaging platforms.