Medical information and correlative histology
Before recording tomographic scans, tissue sections of 2.5⁢μ⁢m thickness were cut from the top, stained by HE (hematoxylin and eosin) and imaged with a microscope. Appendix 1—figure 1 shows the histological slice of each sample. The imaged section is just above the upper plane of the 3D PC-CT reconstruction volume, but not part of it.
Appendix 1—figure 1.  Microscopic images of HE-stained histological sections of all samples (I–VI).
Histological slices show comparable morphologies to the virtual slices in Figure 3, which represent different z-position. Scale bars: 400⁢μ⁢m. An overview of different morphological features identified by conventional HE histology and virtual 3D histology is presented in Appendix 1—figure 2, while Appendix 1—figure 3 presents a direct comparison for the same slice. For this purpose, the sample was sectioned and stained after the PC-CT scan. Artery lumen, artery wall, erythrocytes, thrombus, alveolar septum, marcophage, hyaline membrane and black granules (anthracosis) are shown in Appendix 1—figure 2 for both imaging methods. Contrast of the hyaline membrane is homogenous in both modalities, facilitating idetification and segmentation. Erythrocytes are easily recognized by eye in the conventional histology image, due to the HE staining, but less well distinguished by virtual histology. This results in a difficult differentiation between thrombus and blood stasis as well as a difficult identification of blood capillaries in the alveolar septum. Importantly, however, feature identification can be confirmed by correlative 2d and 3D histology on the same section, as exemplified by Appendix 1—figure 3.
Appendix 1—figure 2.  Comparison of morphological features between conventional HE histology and virtual histology.
Artery lumen, artery wall, erythrocytes, thrombus, alveolar septum, macrophage, hyaline membrane and anthracotic pigments (i.e. the black granules) are presented on exemplary slices of different samples (I–VI) for conventional (left) and virtual histology (right). Scale bars: left 200⁢μ⁢m, right 100⁢μ⁢m.
Appendix 1—figure 3.  Direct comparison of virtual and HE histology for an identical slice/section.
(top left) Region of interest of the parallel beam tomogram (Sample II). (bottom left) Corresponding HE stained histology slice. Thrombi and erythrocytes can be identified in both imaging modalities. The red square marks the position of the zoom tomogram, for which the corresponding slice is shown on the right. Scale bars: 50⁢μ⁢m.