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Edwards S, Meineke B, Bauer S, Blom H, Elsässer S, Brismar H
Nano Lett. - (-) - [2026-01-22; online 2026-01-22]
With recent advances in fluorescence microscopy, resolution is often limited by the size of the label and the resulting linkage error, rather than the microscope itself. Site-specific incorporation of noncanonical amino acids (ncAAs) combined with bioorthogonal click chemistry provides a powerful tool for fluorescent protein labeling, overcoming the spatial uncertainty inherent to antibody-based probes. Here, we present a method to further improve labeling precision by combining ncAA labeling with expansion microscopy (ExM) for dual-color super-resolution imaging. After optimizing labeling procedures and fluorophore selection, we visualize and resolve the nanoscale distribution of Na,K-ATPase α1 and β1 subunits in expanded HEK 293T cells. We validate our approach by super-resolution STED imaging of the ncAA labeled β1 subunit in unexpanded cells. This work presents a strong framework for multiplexed, high-resolution imaging, suggesting that ncAA labeling combined with ExM enables biological imaging at the nanometer scale.
Integrated Microscopy Technologies Stockholm [Technology development]
PubMed 41571281
DOI 10.1021/acs.nanolett.5c05301
Crossref 10.1021/acs.nanolett.5c05301