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Dual-Color Expansion Microscopy of Membrane Proteins Using Bioorthogonal Labeling
KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-7930-7977
Science for Life Laboratory, Karolinska Institute, 171 21 Solna, Sweden.
Science for Life Laboratory, Karolinska Institute, 171 21 Solna, Sweden.
KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics.ORCID iD: 0000-0002-5584-9170
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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

Available from: 2026-02-20 Created: 2026-02-20 Last updated: 2026-02-20Bibliographically approved

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Edwards, StevenBlom, HansBrismar, Hjalmar

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