Infrared microscopic imaging of bone: Spatial distribution of CO32-

This article describes a novel technology for quantitative determination of the spatial distribution of CO32- substitution in bone mineral using infra red (IR) imaging at similar to \6 mum spatial resolution. This novel techno logy consists of an IR array detector of 64 x 64 elements mapped to a 400 m um x 400 mum 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 var iation of any IR parameter across the array may be mapped. In the current s tudy. a linear relationship n as observed between the band area or the peak height ratio of the CO32- v, contour at 1415 cm(-1) to the PO43- v(1),v(3) contour in a series of synthetic carbonated apatites, The correlation coef ficient between the spectroscopically and analytically determined ratios (R -2 = 0.989) attests to the practical utility of this IR area ratio for dete rmination of bone CO32- levels. The relationship forms the basis for the de termination of CO32- in tissue sections using IR imaging. In four images of trabecular bone the average CO32- 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 h ighest levels of CO32- were found at the edge of the trabeculae and immedia tely adjacent to the Haversian canal. Examination of parameters derived fro m the phosphate v(1), v(3) contour of the synthetic apatites revealed that the crystallinity/perfection of the hydroxyapatite (HA) crystals was dimini shed as CO32- levels increased. The methodology described will permit evalu ation of the spatial distribution of CO32- levels in diseased and normal mi neralized tissues.