Monitoring the size and lateral dynamics of ErbB1 enriched membrane domains through live cell plasmon coupling microscopy.

To illuminate the role of the spatial organization of the epidermal growth factor receptor (ErbB1) in signal transduction quantitative information about the receptor topography on the cell surface, ideally on living cells and in real time, are required. We demonstrate that plasmon coupling microscopy (PCM) enables to detect, size, and track individual membrane domains enriched in ErbB1 with high temporal resolution. We used a dendrimer enhanced labeling strategy to label ErbB1 receptors on epidermoid carcinoma cells (A431) with 60 nm Au nanoparticle (NP) immunolabels under physiological conditions at 37°C. The statistical analysis of the spatial NP distribution on the cell surface in the scanning electron microscope (SEM) confirmed a clustering of the NP labels consistent with a heterogeneous distribution of ErbB1 in the plasma membrane. Spectral shifts in the scattering response of clustered NPs facilitated the detection and sizing of individual NP clusters on living cells in solution in an optical microscope. We tracked the lateral diffusion of individual clusters at a frame rate of 200 frames/s while simultaneously monitoring the configurational dynamics of the clusters. Structural information about the NP clusters in their membrane confinements were obtained through analysis of the electromagnetic coupling of the co-confined NP labels through polarization resolved PCM. Our studies show that the ErbB1 receptor is enriched in membrane domains with typical diameters in the range between 60-250 nm. These membrane domains exhibit a slow lateral diffusion with a diffusion coefficient of D = |0.0054±0.0064| µm(2)/s, which is almost an order of magnitude slower than the mean diffusion coefficient of individual NP tagged ErbB1 receptors under identical conditions.