Dual-Color Expansion Microscopy of Membrane Proteins Using Bioorthogonal LabelingShow others and affiliations
2026 (English)In: Nano Letters, ISSN 1530-6984, E-ISSN 1530-6992, Vol. 26, no 4, p. 1321-1326Article in journal (Refereed) Published
Abstract [en]
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
Place, publisher, year, edition, pages
American Chemical Society (ACS) , 2026. Vol. 26, no 4, p. 1321-1326
Keywords [en]
Bioorthogonal chemistry, Expansion microscopy, Linkage error, Noncanonical amino acids, Site-specific labeling, Super-resolution microscopy
National Category
Molecular Biology Cell and Molecular Biology Biophysics Cell Biology
Identifiers
URN: urn:nbn:se:kth:diva-376992DOI: 10.1021/acs.nanolett.5c05301ISI: 001668757300001PubMedID: 41571281Scopus ID: 2-s2.0-105029189485OAI: oai:DiVA.org:kth-376992DiVA, id: diva2:2040417
Note
QC 20260220
2026-02-202026-02-202026-02-20Bibliographically approved