Fluorescence microscopy is a commonly employed technique for examining cell-matrix interactions. Fluorophores, including fluorescent fusion proteins, have been used for studying ECM remodeling; cell adhesions are often studied using total internal reflection fluorescence microscopy (TIRFM). We show here that as an alternative, SPRI can be a sensitive, label-free, and low-light optical method that eliminates the requirement for modified biological molecules and transfected cells, and allows for real time observation of protein deposition and live cell engagement with the surface.
Surface plasmon resonance (SPR) is an evanescent wave refractive index measurement made at a metal-coated surface and has been demonstrated to be a quantitative, sensitive, and label-free technique for measuring the binding kinetics of proteins, DNA, and small molecules to surface-immobilized capture agents.
We demonstrate that SPRI contrast allows sensitive and accurate quantification of cell areas and dynamic changes in cell-substrate interactions such as membrane ruffling. SPRI also allows quantification of secretion of proteins from cells, and changes in surface protein density as a function of time and location. The SPRI apparatus used in this study is similar to literature reported SPR imaging configurations but the SPR sensor surface is positioned horizontally and incident light is launched into a gold-coated SF-10 glass slide which comprises the cell culture surface of an enclosed chamber. The cell chamber can be easily transferred between the SPR imaging apparatus and an inverted optical microscope.
The figure shows an SPR image (right) of vSMC collected using 470 nm incident light, and corresponding phase contrast (left) and fluorescence (center) images for the same field of view. The fluorescence image was produced by staining fixed cells with Texas Red maleimide. The surface was prepared by using microcontact printing to apply fibronectin to the surface in squares of 300 μm by 300 μm, separated by areas of (PEG)-terminated alkanethiol (PEG-thiol) to produce areas resistant to protein adsorption. Only the SPR image allows visualization of both the protein patterned regions as well as the cell-substrate contacts.
Image analysis techniques, such as object segmentation, are used to extract quantitative information from the SPR images. For example, live-cell measurements by SPRI are used to measure the rates of cell spreading and lateral ruffling. Cells can be observed over long periods of time because cellular damage is minimized at the low light levels that are sufficient for SPRI. Quantitative measurement of cell-derived protein was analyzed by image analysis of protein deposited as a function of proximity to cell edge. We detected as much as 120 ng/cm² of protein deposited by cells in 24 h.