Dark Field Colored Imaging of Rat Bone Shows the Variation of Nanocrystal Orientation Within and Between Mineral Platelets

Bone is comprised of crystals of calcium phosphate surrounding collagen fibrils. The crystals occur in polycrystalline mineral platelets (MPs). The c-axes of the nanocrystal components of the platelets have an overall preferred orientation parallel to the collagen fibrils but which varies somewhat. In this contribution we show how dark-field (DF) image processing can be used to measure and visualize nanocrystal orientations over micrometer-sized areas. We recorded selected area electron diffraction patterns from ion-beam milled TEM sections of the femora of Wistar rats cut parallel to the collagen fibrils. Diffraction arcs of the 002 reflection represent the variation in the orientation of c-axes of the nanocrystals. Performing DF imaging of electrons from five consecutive, discrete portions along these arcs, we created a series of images. Using a different color coding for each portion, we constructed a merged DF image, which allows intuitive visualization of gradual change of nanocrystal orientation. We show that many complete platelets are built of crystals whose c-axes vary in orientation by up to 26°. In some regions, stacks of platelets have essentially uniform c-axis orientation. These could be “tesselles” which make up only small fraction of volume of bone. Elsewhere adjacent MPs may differ widely (up to 50°) in orientation.