In particular, a threshold for the minimal area Am of macrophages on the red layer (split point 3 in Fig. 1) Y-27632 solubility dmso and thresholds for the minimal areas As and Acs of single spores and clustered spores, respectively, were used on the green layer (split points 4–6 in Fig. 1). We used different thresholds for single spores and clustered spores, Acs < As, because largely overlapping fluorescence signals in the images appear for spores that are lying close together in clusters. Furthermore, to distinguish spores from artifacts in the images, thresholds for object roundness and object asymmetry were used in addition to the area feature (split

points 5 and 6 in Fig. 1). Here, object roundness was evaluated by approximating the ROI by an outer and inner ellipse and Raf inhibitor by computing the difference σ between the major axis of the outer ellipse and the minor axis of the inner

ellipse.[16] In contrast, the object asymmetry was computed from the ratio of the main axes rmax and rmin of an ellipse that was fitted to the ROI as α = 1 − rmin/rmax. Here, we distinguished again between thresholds for the roundness of single spores σss and clustered spores σcs, and similar for the asymmetry of single spores with threshold αss. We modified the implemented algorithm[16] to deal with the current image data by dividing the segmentation into two sub-steps. Here, we first computed for each image an intensity threshold automatically and then applied the multi-threshold segmentation algorithm. With regard to the size of the spores (see split points 4–6 in Fig. 1), we enforced only a lower but not

an upper threshold and by that enhanced the probability of detecting all spores to ensure that the number of missed spores was minimal, i.e. we were opting for a high recall. However, since this segmentation sub-step did not distinguish Acyl CoA dehydrogenase between ROIs that are single spores or clustered spores, a second segmentation sub-step was required where clusters of spores were split into single spores based on the features roundness and asymmetry. The ruleset distinguishes between phagocytosed and non-phagocytosed spores being adherent and non-adherent to macrophages (split point 7 and 8 in Fig. 1). The decision of the class memberships for spores was made on the blue layer, because due to the staining only adherent and non-adherent spores that were not phagocytosed appear in blue. ROIs are classified as spores or artifacts in the images depending on their average intensity I relative to the threshold value Is in the range of integer values between 0 and 255. We optimised the value of Is (see Table 1) by a validation procedure involving a manual classification on selected images. Finally, non-phagocytosed spores were classified as adherent or non-adherent to macrophages (split point 8 in Fig. 1) depending on whether or not they share a border with macrophages on the red layer.