Fig. 1.

a) In the background: overview image of part of the femoral cross-section. Stained in red by rhodamine are the Haversian canals, the osteocyte lacunae and the canaliculi. b) Stack of images obtained by confocal laser scanning microscopy (CLSM) used to render the three-dimensional topology of the OLCN. c) Visualization of a small subvolume to demonstrate the steps in the image analysis: the three-dimensional CLSM image is thresholded to obtain the red contour surface. Subsequent skeletonization and fitting with smoothing splines (dark lines) provides a mathematical rendering of the network topology. A segmented lacuna is visualized in purple. In d) the network of a representative dataset is visualized: osteocyte lacunae (blue), Haversian canal in the center of the osteon (green) and skeletonized canaliculi, which are color coded to indicate their distance from the Haversian canal (from blue to red). The subvolumes of 400 μm³ used to assess the heterogeneity of the canalicular density are of equal size than medium osteocyte lacunae. The cylindrical coordinate system used in the evaluation of the canalicular density is sketched in e).