Dual-color expansion microscopy of membrane proteins using bioorthogonal labelling

Site-specific incorporation of non-canonical amino acids (ncAAs) combined with bioorthogonal click chemistry provides a powerful tool for fluorescent protein labeling, overcoming the linkage error inherent to antibody-based probes. In this study, we present the development of dual-color super-resolution imaging utilizing ncAA labeling together with expansion microscopy (ExM). After optimizing the labeling procedures and fluorophore selection, we visualize and resolve the nanoscale distribution of Na,K-ATPase 1 and {beta}1 subunits in expanded HEK 293T cells. We validate our approach by super-resolution STED imaging of ncAA labeled {beta}1 subunit in unexpanded cells. This work establishes a robust framework for multiplexed, high-resolution imaging and suggests that the combination of ncAA labeling with ExM has the potential to push biological imaging toward [A]ngstrom-level resolution.