Fig. 4.

3D volumetric reconstruction of BSE micrographs obtained via serial block-face SEM. a Matrix surrounding cells in non-mineralized collagen had little backscattered contrast, suggestive of lack of mineralization. b In mineralized hydrogels, the matrix was visibly darker due to the backscattered electron contrast of mineralized fibrils, especially in the matrix immediately surrounding the cells. c Collagen in OIM-treated samples also lacked significant backscattered electron signal. d Illustration of the serial stacking of 190 60 nm-thin sections, the segmentation of cells (blue) from the surrounding mineralized matrix (middle panel, scale bar: 20 µm), and visualization of block 3D image (right panel). Arrows in d show narrow dendrite-like cell processes. e 3D-rendered image of mineralized samples showing cells (blue) embedded in mineral (red), with the underlying collagen (gray). f Exclusion of collagen via digital processing in these mineralized samples illustrates the density of mineralized collagen and cells spread within a bed of mineralized matrix. Narrow cell processes (arrows) shown in higher magnification in g appear to extend between mineralized fibrils (Supplementary Movie 2) (scale bar: 10 µm). h Digital removal of cell bodies from within the mineralized matrix illustrates density of mineral surrounding the cell structures. The total length of the x-axis in all 3D reconstructions (d, e, f, and h) is 62 µm