Using Surface Plasmon Resonance Imaging to Probe Dynamic Interactions Between Cells and

Abstract Spatially resolved details of the interactions of cells with a fibronectin modified sur-face were examined using surface plasmon resonance imaging (SPRI). SPRI is a label-free technique that is based on the spatial measurement of interfacial refractive index.SPRI is sensitive to short range interactions between cells and their substratum.The high contrast in SPR signal between cell edges and substratum facilitates identifi-cation of cell edges and segmentation of cell areas. With this novel technique,we demonstrate visualization of cell-substratum interactions, and how cell-substra-tum interactions change over time as cells spread, migrate, and undergo membraneruffling. Published 2010 Wiley-Liss, Inc. y Key terms vascular smooth muscle cells; fibronectin; imaging; image analysis; label-free; surfaceplasmon resonance; cell spreading; ruffling T HE extracellular matrix (ECM) is primarily composed of insoluble proteins sur-rounding cells, which serves to provide mechanical support and anchorage for cellsas well as specific chemical and mechanical cues. The ECM influences intracellularsignaling pathways and cytoskeletal organization, and is involved in regulating a cell’sdynamic behavior (1–3). A critical challenge in cell biology is tracking and quantify-ing the interactions of cells with the ECM.Direct visualization and measurement of cell-ECM interactions are possible usinga limited numberofoptical techniques suchastotal internalreflection fluorescence mi-croscopy (TIRFM). TIRFM is a fluorescent-based measurement that employs an eva-nescent wave of excitation light to selectively excite regions of cell-substratum contact.It requires fluorescent labeling of the cell-membrane (4), the cell cytosol (5), or theECM proteins. Interference reflection microscopy allows label-free measurement ofcell-substratum interaction, but the required optical architecture limits the field ofview to small, single-cell regions, and the complexoptical response precludes quantita-tion of mass or molecular density (6). Optical waveguides have been used as a label-free measurement technique to quantify cell spreading (7) and is sensitive to cell-secreted materials (8), but it does not provide visualization of cellular features.Here we demonstrate the use of surface plasmon resonance imaging (SPRI) tospatially resolve and quantitatively analyze both the ECM proteins and the dynamicbehavior of live cells. Surface plasmon resonance (SPR) is a label-free evanescentwave technique that is essentially a highly sensitive refractive index measurementnear a metallic surface (9). In a nonimaging mode, SPR spectroscopy has beendemonstrated as a highly useful and sensitive technique for quantitative determina-tions of time-dependent changes of binding of proteins (10) and DNA (11) to surfaceimmobilized capture agents. It has been reported that even changes in the dielectric