Image segmentation and quantification
For each patient the stitched overview scans were analyzed with regard to structural characteristics. The 3D-reconstructions were first binned (2 × 2 × 2), and the tissue was then segmented from the surrounding paraffin using the segmentation software Ilastik (Berg et al., 2019), which was then further refined with MATLAB. In order to exclude single macrophages or detached tissue, only voxels connected to the tissue block were considered for the distance map. Further, the areas of the paraffin which represent air compartments were linked to the outside of the tissue block. Individual self-contained areas of paraffin (not connected to air) were excluded from the mask. Based on this segmentation, the distance to the nearest voxel containing oxygen was calculated for each tissue voxel. The tissue volume V is given by the sum of all voxels containing tissue. The surface area SA is defined by all tissue voxels with a distance of 1 pixel to the air. From this information we calculated the specific surface(2) SV=SA/Vwith S⁢A/V surface area volume -ratio. Further a characteristic length(3) Lc=V/SA⋅vxwith v⁢x edge length of a voxel was determined for each sample. Additionally, the mean distance (dO2¯) from all tissue voxels to the closest air compartment and its standard deviation was calculated.
Hyaline membranes and capillary networks were extracted using semi-manual segmentation functions in Avizo (Thermo Fisher Scientific, USA). Beside hyaline membranes and the capillary network, different cell types can be readily identified based on the 3D reconstructions of the electron density. In particular, inflammatory cell subpopulations - that is, macrophages and lymphocytes - can be distinguished. To this end, an automatic and parameter-controlled algorithm denoted as BlobFinder was used (arivis AG, Germany). Based on its segmentation output, the amount and position of the lymphocytes in the 3D-reconstructions from parallel beam scans was calculated. The algorithm is able to identify roundish structures with a given size. For the segmentation of the lymphocytes, we chose a characteristic size of 6.5⁢μ⁢m. The structures identified in this step also include macrophages and parts of the capillary system. For the unbinned datasets, a distinction between lymphocytes and the nuclei of the macrophages was made based on the difference in electron density. In the tomographic reconstructions, the lymphocytes appear denser compared to the nuclei of the macrophages. Further, nuclei from endothelial cells and parts of the capillaries filled with blood residues were excluded based on their elongated shape. Only structures with a sphericity higher than 0.55 were included. Based on the segmentation of lymphocytes, the total number of lymphocytes Nl was obtained and the mean concentration of lymphocytes within the lung tissue cl=Nl/V was estimated.