Understanding the Origin of Surface Plasmon Resonance Microscopy Signals in Cell-Based Molecular Interaction Measurements

Surface plasmon resonance microscopy (SPRM) is widely used for label-free imaging of biomolecular interactions at cell membranes. In cell-based assays, SPRM often produces distinctive patterns, such as edge-dominated signals and spatially heterogeneous binding kinetics, yet the physical origins of these features remain incompletely understood and cannot be resolved by SPRM alone. Here, we combine SPRM with depth- and time-resolved confocal fluorescence imaging to address this question. Using lectin binding on fixed cells as a model system, we show that SPRM signals primarily originate from ligand binding within the portion of the membrane exposed to the evanescent field, with additional contributions from binding-induced membrane deformation. We also find that local membrane features strongly correlate with kinetic heterogeneity. These results provide direct experimental evidence clarifying SPRM signal formation in cell-based measurements and offer practical guidance for accurate quantification and interpretation in label-free plasmonic imaging of cellular interfaces.