MEASURING DENSITY IMMUNOGOLD LABELLING
After immunogold staining (Protein A-gold IGS), the binding of primary antibody can be observed as dense "black grains” in a TEM image. The degree of binding of the primary antibody is reflected by the density of these black grains in the image. Qualifying the density and thus quantifying the degree of binding "de visu" is very difficult and highly arbitrary. Using digital image editing techniques, a fast and precise quantification is possible. The black granules were selected and isolated from a predefined, “clean” area A (= W * H) (= number of pixels width * number of pixels height) in the grey image, using the Photoshop “threshold” function. This interactive feature converts a greyscale (or colour) image into a black-and-white image with sufficient contrast. Hereby, a certain level can be specified as threshold (the threshold value is somewhere in the region of 45-70; the amount is visually adjustable). Doing so, all pixels (picture elements) lighter than this threshold value are converted into white, all pixels darker than the threshold value are converted into black. Following, the degree of binding was expressed as the number N of black pixel clusters, or granules, in the image per unit area (nm² or µm²) using Photoshop Measurement Log (also other counting editing techniques can be used). The scaling factor F, measured from the image scale bar length L as number of pixels P, was used to calculate the image area. The settings of the density number calculus were: Scaling factor F = L / P (nm units); Selected image area A = W * F * H * F (nm² units); Density number D = N / A (1/nm² units) or N / A * 106 (1/µm² units).