Multiplexed Lectin-PAINT super-resolution microscopy enables cell glycotyping
2025
Marrit M. E. Tholen | Roger Riera | Cristina Izquierdo-Lozano | Lorenzo Albertazzi
Abstract Glycosylation profoundly influences cellular function, yet deciphering its intricate patterns remains a formidable challenge. Current techniques often compromise sensitivity, multiplexing, or the ability to capture in-situ cell-to-cell variations. To address these limitations, we introduce ‘Lectin-PAINT,’ a super-resolution imaging method enabling multiplexed live-cell visualization of the cellular glycocalyx at the single-cell and single-molecule levels. Lectin-PAINT leverages the reversible binding of lectins to specific carbohydrate families to perform point accumulation in nanoscale topography (PAINT), enabling the identification, mapping, and tracking of carbohydrates with a resolution beyond the diffraction limit. Our technique harnesses a tailored lectin library, spanning key carbohydrate recognition, offering insights into their abundance, affinity, and mobility. Through 8-color super-resolution imaging, we extract more than 350 glycosylation parameters with single-cell resolution, creating a cell’s ‘glycotype’ or glycan fingerprint. We showcase the power of this approach by glycotyping and categorizing a diverse set of cancer cell types, shedding light on the heterogeneity and variability of the glycocalyx in cancer. In the future, this research will contribute to the more fundamental understanding of changes in the glycocalyx due to disease.
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