For SBF-SEM an image stack of a bovine placenta sample was used. The sample was collected from a placentome of a pregnant Holstein Friesian cow (gd 278 days), terminated by cesarean section, which was performed after rupture of fetal membranes. Preservation in Karnovsky’s fixative was followed by embedding in Durcupan’s resin, as previously described [56 ]. These were recorded with a section thickness of 60 nm. SBF-SEM images were used for further processing in 3D reconstruction and modeling of the target cell type.
ROIs with well-preserved areas of maternal stroma were selected on single SEM surface scans of the block. The selected image field, showing the target cells, was utilized for the creation of an image stack with 970 images. 3D reconstruction was performed on a defined stack of 180 images (Voxel size = 15 × 15 × 60 nm), which included a complete myofibroblast. Herewith, a manual segmentation of the nucleus, the stress fibers and the cytoplasm, was conducted, using the ImageJ software (ImageJ 1.53q, bundled with 64-bit Java, version 1.8.0_172) plugin TrakEM2 [57 (link)]. For visualization, segmented images were merged and resulting channels were used to create a 3D model (Imaris ×64, version 9.9.1, Bitplane AG).
ROIs with well-preserved areas of maternal stroma were selected on single SEM surface scans of the block. The selected image field, showing the target cells, was utilized for the creation of an image stack with 970 images. 3D reconstruction was performed on a defined stack of 180 images (Voxel size = 15 × 15 × 60 nm), which included a complete myofibroblast. Herewith, a manual segmentation of the nucleus, the stress fibers and the cytoplasm, was conducted, using the ImageJ software (ImageJ 1.53q, bundled with 64-bit Java, version 1.8.0_172) plugin TrakEM2 [57 (link)]. For visualization, segmented images were merged and resulting channels were used to create a 3D model (Imaris ×64, version 9.9.1, Bitplane AG).
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