Illustration of the image analysis procedure to quantify protein deposition at cell edge. A) A histogram of image reflectivity pseudocolored with the colorbar scale shown is divided into three distinct regions: PEG-thiol (dark blue), fibronectin/protein (light blue-green), and cell features (yellow-red). B) A line that spans the regions of cell contact, fibronectin/protein layer, and PEG-thiol layer is shown as the grey bar in (C) and the reflectivity values under that line is shown in (B). The horizontal blue bars mark the width of the transition (average 5 μm to 8 μm) from one region to the other. C) Sequential image analysis steps for a SPR topographic image of a 300 μm square fibronectin island where the z-axis corresponding to reflectivity values shown in the colorbar. The first image shows the cell, protein, and PEG-thiol regions with a black ring segmenting the cell-protein threshold, at a reflectivity value of 0.20, as displayed in (A) and (B). The second image deletes the cell objects and the black line highlights the initial cell contour ring that still borders part of the cell edge as seen in (B). The third image dilates the cell contour by 5 pixels clearing the cell-protein transition region as shown in (B). It is at this point that sequential contour dilating to determine surface protein coverage surrounding cells is begun. The black scale bar equals 300 μm, and the gray bar corresponds to the line scan used in (B).
Peterson et al. BMC Cell Biology 2009 10:16 doi:10.1186/1471-2121-10-16