Abstract
Spatial clustering of cell membrane receptors has been indicated to play a regulatory role in signal initiation, and the distribution of receptors on the cell surface may represent a potential biomarker. To realize its potential for diagnostic purposes, scalable assays capable of mapping spatial receptor heterogeneity with high throughput are needed. In this work, we use gold nanoparticle (NP) labels with an average diameter of 72.17 ± 2.16 nm as bright markers for large-scale epidermal growth factor receptor (EGFR) clustering in hyperspectral plasmon coupling microscopy and compare the obtained clustering maps with those obtained through fluorescence superresolution microscopy (direct stochastic optical reconstruction microscopy, dSTORM). Our dSTORM experiments reveal average EGFR cluster sizes of 172 ± 99 and 150 ± 90 nm for MDA-MB-468 and HeLa, respectively. The cluster sizes decrease after EGFR activation. Hyperspectral imaging of the NP labels shows that differences in the EGFR cluster sizes are accompanied by differences in the average separations between electromagnetically coupled NPs. Because of the distance dependence of plasmon coupling, changes in the average interparticle separation result in significant spectral shifts. For the experimental conditions investigated in this work, hyperspectral plasmon coupling microscopy of NP labels identified the same trends in large-scale EGFR clustering as dSTORM, but the NP imaging approach provided the information in a fraction of the time. Both dSTORM and hyperspectral plasmon coupling microscopy confirm the cortical actin network as one structural component that determines the average size of EGFR clusters.