Figure 5

Quantitation of protein deposition using SPRI. A) Representative SPR images (using 470 nm incident light) of individual 300 μm squares of fibronectin for regions of relatively high and low cell density. Fibronectin areas are separated by areas of PEG which are resistant to protein adsorption. Image contrast is optimized to aid visualization of differences in protein deposition. The intensity values correspond to reflectivity values shown in the scale bar; reflectivity values > 0.200 appear white and < 0.165 appear black. To determine the amount of protein added to the surface during 24 h in the presence of cells, a 100 μm square region of interest (magenta box) adjacent to cells was selected, and SPR reflectivity within the blue box was averaged and converted to mass/area. A similar region was selected from a region with no cells. B) Bar graphs show that mass of material added to the surface at different steps, and in areas largely devoid of cells and areas containing cells. Mean values and standard deviations are the result from 5 imaged areas. C) Image analysis is used to quantify protein deposition at the cell periphery after 24 h in culture by dilating the cell contours to extract protein mass versus distance from the cell edge in a region where 18% of the area is occupied by cells (approximately 25 cells per field of view). Sequential dilation operations are performed in 1-μm increments. The magenta-colored contours overlay fibronectin regions; green contours overlay PEG-thiol regions. The lines shown around the cells correspond to a distance of 40 μm from the cell edges. Protein coverage in ng/cm² is determined from the average reflectivity values for each sequential contour. Protein versus distance from cell edge is shown for fibronectin-coated regions (D). The corresponding plot in green is for the PEG-thiol regions (E). The error bars represent the standard deviation from 4 different sample regions. Scale bars = 100 μm. See text for a more complete description of the collection of these data.