Segmentation of the fixed structure.

<p>The color image (a) is segmented by relative RGB color planes (b, c). Some morphological operations (scrapping of small objects, closing) yield the blue stained area A_B (d).</p

Segmentation of the mobile cells.

<p>(In this example: CD56⁺ cells). The color image (a) is segmented by relative RGB color planes (b, c). Some morphological operations (closing, opening) yield the red stained area A_R (d).</p

Results of analysis of samples.

<p>(a) Relative density of CXCR3⁺ cells in neighborhood of capillaries and venules. (b) Relative density of CD56⁺ cells in neighborhood of capillaries and venules. (c) Relative density of CD56⁺ cells in neighborhood of spiral arteries. The data are individually normalized in relation to density in residual decidua = 1. (y axis, log scale.)</p

Quantile - quantile plot for neighborhood 10 µm.

<p>The dots (red: CXCR3⁺ cells in relation to endothelium, green: CD56⁺ cells in relation to endothelium, blue: CD56⁺ cells in relation to the tunica media of spiral arteries) represent the medians of patients' data of cell densities in the neighborhood (x-axis) versus in the residual decidua (y-axis). The black line represents the random distribution, the black bars the standard deviation of the random distribution.</p

Definition of the endothelial neighborhood by morphological dilation.

<p>The segmented image of the endothelium (a) is morphologically dilated (b). (Here a neighborhood width of 30 µm is chosen.) The dilated image A_B + N (c) subtracted with the blue stained endothelial area A_B (d) yields the endothelial neighborhood area A_N (e). The total specimen area A_T (f) subtracted by the dilated area A_B + N (g) yields the residual decidua area A_D (h). A_N: Neighborhood area, A_D: Area of residual decidua, A_B: Area of endothelium, A_T: Area of total specimen.</p

Examples of microphotographs for automated analysis: Two-color immunohistochemistry of non-invaded first trimester decidua.

<p>(a) Red: (mostly uterine NK) cells stained with anti-CD56-HRP-AEC, blue: CD34⁺ vascular endothelium stained with anti-CD34-APAAP-NBT/BCIP. (b) Red: CD56⁺ cells, blue: tunica media of spiral arteries stained with anti-smooth muscle actin – APAAP-BCIP.</p

Endothelial neighborhood (A_N) and area of residual decidua (A_D) with increasing neighborhood width.

<p>Endothelial neighborhood (A_N) and area of residual decidua (A_D) with increasing neighborhood width.</p

Artificial images with randomly scattered cell distributions.

<p>For the sake of better comparison of cell distributions at different overall cell densities a model of random distribution of artificial cells in relation to neighborhood areas and rest decidua of a specific sample was generated. Randomly distributed cells projected onto a sample object of the total specimen's area A_T (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015086#pone-0015086-g003" target="_blank">Figure 3b</a>). (a) Low cell density with 100 seeds, (b) high cell density with 500 seeds.</p

Definition of neighborhood area on the basis of a microphotograph of two-color immunohistochemistry of first trimester decidua (see Figure 1a).

<p>Generation of an area of vicinity of a size to be chosen (in our example, 3 sizes at distances of up to 10, 20, and 30 µm from the vascular endothelium are given).</p