4.6. Digital Image Analysis
Slides were scanned as high-resolution whole-slide images at a resolution of 0.17 µm per pixel (SlideStrider scanner, Jilab Inc., Tampere, Finland) with three alternative illuminations. First, brightfield illumination (for hematoxylin counterstaining) was used to define the tissue boundaries and the focus plane and to provide the histologic context of the biopsy. The second image layer was the scan with green-emitting fluorescence light, and the third was for capturing the deep red-emitting fluorescence. The image layers were saved in the multilayer JPEG2000 image format and viewed with the SlideVantage whole-slide viewing system (Jilab). SlideVantage allows viewing each layer separately or blended to reveal fluorescence co-localization.For scoring the staining intensity, we developed a quantitative digital image analysis algorithm to measure the mean integrated fluorescence signal intensities of the enterocyte or lamina propria tissue areas defined by the user (