Figure 3

Digital image analysis; work flow. Representative raw and digitalized images of a rectal tissue section stained for E-Cadherin (green), CD4 (red) and DAPI (blue) illustrating the workflow of our customized digital image analysis. (a) A raw fluorescent image of DAPI staining (blue, first column) was used for detection of cells, shown as outlines in the second and third column. Cells were detected using CellProfiler, and the software program Ilastik was used to classify cells as either epithelial cells (second column) or lamina propria (LP) cells (third column). The digitalized images in the fourth column are magnified views of the regions indicated in the second and third columns (epithelial cells upper image and LP cells lower image). Likewise, raw images, in the first column, of immunoflourescent staining of (b) E-cadherin (green), (c) CD4 (red) and (d) E-cadherin, CD4 and DAPI. Images in the second column are digitalized images (exported from CellProfiler and colorized in Matlab) representing analysis results of the images in the first column. The raw- and digitalized images in the third column are magnified views of the regions indicated in the first and second column, respectively. The following color codes were used in (d) E-cadherin positive epithelial cells in green, E-cadherin negative epithelial cells in dark grey, CD4 positive cells in red and CD4 negative submucosal cells in beige.