Segmentation and volume quantification of villous tissue and vessels
Images were pre-processed, analysed, and viewed withFIJI 26. Pre-processing consisted of de-noising with a 3D median filter, sigma 1. Subsequently, placental villi (autofluorescence), vessels, and SHG signal were segmented with the FIJI Labkit plugin27.
Volume and surface areas of vessels and villi were quantified with3D ImageJ suite 28 (Figure S9, S10). Binary images were imported into 3D ROImanager 28 to obtain surface area and volume measurements. Villous vascular fraction was calculated by dividing the vascular- and villous volume and converted to percentage. The surface area to volume ratio (SA/Vol) was quantified as a measure for effective diffusion area.