Cellular remodeling of the ECM is a critical factor in wound healing, developmental biology, metastasis of tumor cells, and diseases such as hypertension [1
]. The study of cell-matrix dynamics and cellular remodeling of the ECM is challenging, and has involved the use of fluorophores, including fluorescent fusion proteins [5
], often using total internal reflection fluorescence microscopy (TIRFM) [6
]. 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 highly sensitive real-time observation of protein deposition and live cell engagement with the ECM.
Surface plasmon resonance (SPR) occurs when light energy couples into the electromagnetic field at a metal-coated surface. The reflectivity of the incident light is inverse to the extent of plasmon resonance, and is determined by the identity and the thickness of the metal layer, the angle of incidence, the wavelength of the incident light, and the refractive index of the medium at the interface. Because the refractive index is proportional to the amount of adsorbate at the surface [7
], SPR has been used as a quantitative, sensitive, and label-free technique for measuring the binding kinetics of proteins [8
], DNA [9
], and small molecules [11
], to surface immobilized capture agents. Using SPR in an imaging mode, high throughput analysis of proteins and DNA has also been demonstrated [13
]. SPR imaging has not previously been considered a useful technique for imaging cell features, largely because of previous assumptions that poor spatial resolution would prevent useful imaging.
In this report, we demonstrate that SPRI contrast allows sensitive measurement of cell-substrate interactions and mass changes at the substrate interface. SPRI allows quantification of cell secreted and deposited material by observing changes in surface protein mass/area as a function of time and location. We use SPRI to observe the initial surface preparation by monitoring the deposition of the extracellular matrix protein fibronectin which serves as the substrate for the cell based measurements. By using different incident wavelengths and image processing routines for SPRI, it is possible to tune the SPRI measurement for sensitivity versus spatial resolution to suit each step of the experiment. In this report, we demonstrate that SPRI is a sensitive interfacial technique that is able to bridge the gap between molecular (protein adsorption) and cellular (cell-substrate) measurements.