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. 2014 Apr 29;24(5):475–493. doi: 10.1111/bpa.12134

Figure 1.

figure

Image processing for distance frequency analysis. For aquaporin 4 (AQP4)–neuronal membrane distance frequency analysis, images captured from slides double stained for AQP4 (red) and nicotinic acetylcholine receptor (AchR) (green) (A) were first processed for extracting and saving the outlines of the AQP4‐positive pixels based on a constant histogram‐based profile on the red channel (B). Neuron masks were also created with the aid of another histogram‐based profile in the green channel, respectively (C), and a three‐dimensional distance map filter algorithm was applied to generate a distance map image (D) where each pixel's intensity represents its distance to the nearest black pixel from the mask itself. The outlines of the AQP4 signal were then overlaid on the distance map image (E), and their mean intensities (representing now the mean distances to the edge of the nearest neuronal outline) were saved for each image. An identical algorithm was utilized for AQP4–vascular basement membranes distance frequency analysis based on the AQP4 (red) and collagen IV (Col. IV) (green) immunofluorescence signals (F–J). Scale bars represent 50 μm.