Infrared microscopic imaging of bone: spatial distribution of CO3(2-)

This article describes a novel technology for quantitative determination of the spatial distribution of CO3(2-) substitution in bone mineral using infrared (IR) imaging at approximately 6 microm spatial resolution. This novel technology consists of an IR array detector of 64 x 64 elements mapped to a 400 microm x 400 microm spot at the focal plane of an IR microscope. During each scan, a complete IR spectrum is acquired from each element in the array. The variation of any IR parameter across the array may be mapped. In the current study, a linear relationship was observed between the band area or the peak height ratio of the CO3(2-) v3 contour at 1415 cm(-1) to the PO4(3-) v1,v3 contour in a series of synthetic carbonated apatites. The correlation coefficient between the spectroscopically and analytically determined ratios (R2 = 0.989) attests to the practical utility of this IR area ratio for determination of bone CO3(2-) levels. The relationship forms the basis for the determination of CO3(2-) in tissue sections using IR imaging. In four images of trabecular bone the average CO3(2-) levels were 5.95 wt% (2298 data points), 6.67% (2040 data points), 6.66% (1176 data points), and 6.73% (2256 data points) with an overall average of 6.38+/-0.14% (7770 data points). The highest levels of CO3(2-) were found at the edge of the trabeculae and immediately adjacent to the Haversian canal. Examination of parameters derived from the phosphate v1,v3 contour of the synthetic apatites revealed that the crystallinity/perfection of the hydroxyapatite (HA) crystals was diminished as CO3(2-) levels increased. The methodology described will permit evaluation of the spatial distribution of CO3(2-) levels in diseased and normal mineralized tissues.