Fig. 3

Comparison of the colocalization via RBF to the standard colocalization analysis in fluorescence microscopy. The golden standard of colocalization analysis in fluorescence microscopy is to analyse the different colour channels (e.g. red and green). In this figure we simply matched this method to IHC by comparing and analysing the “brown” channel obtained by colour deconvolution. One small region of a slide sequentially stained for CD61 (A) and MPO (A) was analysed. Subsequently, colocalization on basis of the herein described RBF was compared to analysis of colour chancels. a One small region with one CD61-positive megakaryocyte (upper image) was compared to the same region shifted one cell width to left (middle image). The upper scatter plot shows the brown colour channels of the images plotted against each other, whereas the lower one shows the scatter plot for the RBF. b The same small region now stained for MPO was used to check for colocalization/direct spatial interaction of the CD61-positive megakaryocyte and the MPO-positive granulocytes (upper image). The arrow in the zoomed part of the image highlight direct spatial interaction of the megakaryocyte and the granulocytes. The upper scatter plot again shows the brown colour channels plotted against each other and the lower one shows the RBFs plotted against each other