Fig. 1

Nonlinear microscopy and morphometric analysis of esophageal lamina propria in pediatric biopsies. a A nonlinear microscope is used to scan b 5 μm thick H&E sections, providing c two-photon excitation fluorescence images (red) and second harmonic generation (SHG) images with collagen fiber contrast (blue). The images are linearly contrast adjusted for visualization (Scale bar: 100 μm). d Backward SHG image depicts the collagen fiber distribution in the lamina propria. e A ridge detection algorithm (Steger method) is used for automated fiber segmentation. f Regions not containing fibers, termed pores (magenta), are extracted using Moore-Neighbor tracing and the white rectangle is magnified in panel g