Identification of the molecular mechanisms in cellular processes that elicit a surface plasmon resonance (SPR) response using simultaneous surface plasmon-enhanced fluorescence (SPEF) microscopy.

Surface plasmon resonance (SPR) has developed into a powerful approach for label-free monitoring of cellular behavior. Most cellular responses, however, involve a complex cascade of molecular events which makes identifying the specific components of cellular behavior dynamics contributing to the aggregate SPR signal problematic. Recently, a number of groups have used surface plasmon-enhanced fluorescence (SPEF) microscopy on living cells. In this work, we show that SPEF microscopy can be used to identify the molecular mechanisms responsible for SPR detection of cellular processes. By specifically labeling the actin cytoskeleton in human epithelial kidney cells (HEK 293) and rat vascular smooth muscle cells (A7r5), we correlate cell reorganization observed in SPEF with SPR signal variations reflecting aggregate cellular changes. HEK 293 cells stimulated with angiotensin-II exhibited transient contraction, appearing as an SPR signal decrease with a subsequent increase above the initial baseline. SPEF micrographs showed a decrease in cellular area followed by actin densification and cell spreading. A7r5 stimulated with Latrunculin A showed actin cytoskeleton depolymerization, generating a steady SPR signal decrease, with SPEF micrographs showing extensive collapse of cell actin structures. We observed that SPR monitoring of cellular response is strongly dependent on minute variations in cellular footprint on the substrate as well as changes in the molecular density in the basal portions of the cells. Therefore, combining SPR with imaging of selective fluorescent markers by SPEF allows a more comprehensive deconvolution of the cellular signal in relation to molecular events within the cells.