Fig 1.
Iliac crest bone marrow stained with hemalum-eosin.
Undecalcified section embedded in Technovit® 9100. The quality of the section is equivalent to a decalcified paraffin section. Note the variable density of haematopoiesis within directly adjacent adipose tissue areas. Scale bar = 100 μm.
Fig 2.
Bone marrow subtractively stained for CD34 (blue) and CD 141 (brown).
CD34 is visualised in small microvesssels (capillaries) and is less intensely expressed in large microvessels (sinuses) which exhibit patchy blue and brown staining. Scale bar = 50 μm.
Fig 3.
The first of 21 serial sections immunostained for CD34 plus CD141 with regions of interest R1 to R4.
Scale bar = 1 mm.
Fig 4.
First scan of R1 (a), R2 (d), R3 (g) and R4 (j) immunostaining in combination with front views of the 3D models coloured for shape diameter (b,e,h,k) and connectivity visualisation (c,f,i,l). In (b), (e), (h) and (k) a shape diameter below 12 μm is depicted in red and above 30 μm in green. A transition from red to green occurs in microvessels of shape diameters from 12 μm to 30 μm. In (c), (i) and (l) the largest continuous microvessel network is depicted in light blue and all smaller networks are green. In (f) there are several large networks coloured light and dark blue. Small structures sized between 7 μm and 28 μm are red in (c), (f), (i) and (l) and all structures with a diameter less than 7μm in any dimension are omitted. (b,c) R1; (e,f) R2; (h,i) R3; (k,l) R4. Scale bar for all images: 100 μm.
Fig 5.
Pipeline of image processing and QC methods, both semi-automatic and manual.
* The quality of registration is commented on in Lobachev et al. [25]. ** 3D rendering does not affect the data.
Fig 6.
(a) R 1 containing 4 small regions for semi-automatic QC of volume data processing. The region visualised in (b) is coloured red. Scale bar = 100 μm. (b) Sequence of QC visualised from left to right in upper and lower part of panel: original scan (1), HSV conversion of scan of current serial section (2), HSV conversion of next serial section (3), interpolation between both serial sections: in this case the fourth of seven images is shown (4), final result of all filtering operations of interpolated images (5), areas different between interpolated images and result of filtering coloured red (6). Scale bar = 25 μm.