A method is described for the construction of three-dimensional infrared images from serial two-dimensional infrared images of various chemical structure parameters acquired from cortical bone. A series of serial cortical bone sections, each 4-5 μm thick, separated by ~100 μm, were microtomed from PMMA-embedded normal human tibia. Twenty-three IR image cubes were acquired from this series, corresponding to an overall sample length of ~2.2 mm. From each image cube, IR images of the spatial distribution of the mineral-to-matrix (protein) ratio, the mineral crystallinity, and the ratio of nonreducible-to-reducible collagen crosslinks were calculated. Two-dimensional images were stacked to form a 3D view of the cortical bone, with contrast provided by variation in the spatial distribution of any of the aforementioned chemical/molecular structural parameters.

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