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bioRxiv · 10.1101/2025.10.10.680277

Dual-color expansion microscopy of membrane proteins using bioorthogonal labelling

Edwards, S.·Meineke, B.·Bauer, S.·Blom, H.·Elsässer, S.·Brismar, H.
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Abstract

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.

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Edwards, S., Meineke, B., Bauer, S., Blom, H., Elsässer, S., Brismar, H.. 2025-10-10. Dual-color expansion microscopy of membrane proteins using bioorthogonal labelling. https://doi.org/10.1101/2025.10.10.680277

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