Exploring human splenic red pulp vasculature in virtual reality. Details of sheathed capillaries and the open capillary network. Supplementary material

Supplementary files demonstrate our data in an interactive application. Please download und unpack the file, then launch file "start.bat". Please note that loading the original images takes some time. If you have a Steam VR headset, it is detected and used automatically.

1. Supplementary file 1
   Localization of arteries and arterioles in ROI 4.
   ROI 4 with all endothelia and some periarterial fibroblasts colored blue (CD34), smooth muscle alpha-actin (SMA) colored white and capillary sheaths depicted in green (CD271). Most arteries and arterioles can be localized by white color. White appears in a vertex of the blue reconstruction of CD34 staining, if a surface reconstructed for SMA labelling is closer than 8.75 µm to this vertex. Capillary sheaths connected to the selected arterioles (marked red in Suppl. file 2) are green and non-connected sheaths fed by other arterioles are dark green (CD271).
2. Supplementary file 2
   Relation of capillary sheaths to terminal arterioles in ROI4.
   ROI 4 with all endothelia and some periarterial fibroblasts colored blue (CD34), selected terminal artery and terminal arterioles manually labelled red and capillary sheaths connected to the labelled arterioles in green (CD271). Non-connected sheaths fed by other arterioles are dark green (CD271). Follicular dendritic cells (FDCs) in follicles are light green (CD271). This file corresponds to Fig. 3 and to Suppl. Video 1-3.
3. Supplementary file 3
   Relation of capillary sheaths to terminal arterioles in ROI 2.
   ROI 2 with all endothelia and some periarterial fibroblasts colored blue (CD34), selected terminal artery and terminal arterioles manually labelled red and capillary sheaths connected to the labelled arterioles in green (CD271). Non-connected sheaths fed by other arterioles are dark green (CD271). FDCs in follicles are light green (CD271). This file corresponds to Fig. 4a,b.
4. Supplementary file 4
   Relation of capillary sheaths to terminal arterioles in ROI 1.
   ROI 1 with all endothelia and some periarterial fibroblasts colored blue (CD34), selected terminal artery and terminal arterioles manually labelled red and capillary sheaths connected to the labelled arterioles in green (CD271). Non-connected sheaths fed by other arterioles are dark green (CD271). FDCs in follicles are light green (CD271)
5. Supplementary file 5
   White pulp in 3D (ROI 3).
   ROI 3 with all endothelia and some periarterial fibroblasts colored blue (CD34) and capillary sheaths in green (CD271). FDCs in follicles are light green (CD271). Arteries and terminal arterioles were not labelled.
6. Supplementary file 6 
   Sheathed capillaries exhibit short open-ended side branches.
   ROI 2 with all endothelia and some periarterial fibroblasts colored blue (CD34) and with selected terminal artery and terminal arterioles labelled light blue. Capillary sheaths connected to the selected arterioles are green (CD271) and unconnected sheaths are dark green (CD271). The open-ended side branches of the selected sheathed capillaries were manually labelled red and are visualized in their entire length. This file corresponds to Fig. 4c.
7. Supplementary file 7
   Terminal arterioles may have a few unsheathed connections to the red pulp capillary network.
   ROI 2 with all endothelia and some periarterial fibroblasts colored blue (CD34) and with selected terminal artery and terminal arterioles labelled light blue. Capillary sheaths connected to the selected arterioles are green (CD271) and unconnected sheaths are dark green (CD271). Connections from a terminal arteriole to the red pulp capillary network lacking sheaths were manually labelled red. A part of the capillary network after the connection is colored yellow. This file corresponds to Figs. 4d,e.
8. Supplementary file 8
   B-lymphocytes accumulate around terminal arteries and in/around capillary sheaths.
   Lower part of ROI 2 with selected terminal artery and terminal arterioles colored blue and connected capillary sheaths in green (CD271). B-lymphocytes were digitally reduced in number and automatically labelled red (CD20). The B-cells accumulate around a terminal artery and around (and inside) capillary sheaths. This file corresponds to Fig. 6 and to Suppl. Video 4.
9. Supplementary file 9
   Reduction of B-lymphocytes is inevitable for visualization.
   ROI 2 showing Suppl. file 8 integrated into the entire ROI 2. A large number of B-lymphocytes is present in the red pulp of ROI 2 (CD20 visualized in white). These cells are self-occluding and necessitate reducing B-lymphocytes as shown in Suppl. Fig. 8 to highlight accumulations.
10. Supplementary file 10
    Red pulp venule with potential connections to capillary network.
    A red pulp venule with capillaries in ROI 1. Capillary and venous endothelium is shown in light blue (CD34). A part of a red pulp venule with potential contributing capillaries (CD34, blue) was manually selected in addition to a terminal arteriole (light red). The capillary sheaths connected with this arteriole are green (CD271). Non-connected sheaths are dark green (CD 271) and FDCs in follicles are light green (CD271). Capillaries from the red pulp network, which appear to be connected to the venule were manually colored red. Two of the red capillaries seem to originate from a sheathed capillary. This file corresponds to Fig. 5a-d and to Suppl. Video 5.

Bounding boxes for all files have a side length of 1 mm with exception of Suppl. File 8 which is 800 (b) x 620 (h) x 614 (d) µm.

As for videos, each video is in H.264 in three quality settings:

• ..._h264_8bit.mov is the most generic HQ setting. Should play everywhere, also with QuickTime.
• ..._h265_10bit.mov is the 10 bit video version with HEVC encoder. Should be the same or higher quality with less size. Might not play everywhere.
• ..._h264_small.mov is the LQ setting with much lower resolution, take this if your download bandwidth is limited.

Now, there are 5 videos to supplement the submission:

1. suppl video 1 - sheath r4 full ... shows the region ROI4 with full vasculature, similar to Fig. 3 of the manuscript,
2. suppl video 2 - sheath r4 select .... shows only the selection of the vasculature from ROI4, similar to Fig. 3 of the manuscript,
3. suppl video 3 - sheath r4 clipping .... showcases the front plane clipping technique we used for diagnostics in VR. This demonstrates the contents of Fig. 8 in dynamics.
4. suppl video 4 - sheath bcell ... shows the selection of the B-cells to supplement Fig. 6 in the manuscript.
5. suppl video 5 - sheath r1 vene ... shows a red pulp venule and its apparent connections, this supplements Fig. 5 of the manuscript.