Aspects of microprobe analysis of mineralized tissues

Mineralizing tissues were the first biological specimens to be examined by the electron microprobe. The necessity for performing analyses at high spatial resolution has led to the increasing use of energy dispersive (ED) detectors in CTEMs/STEMs. Because the ED detector accepts x-rays from a large solid angle, it often detects a large extraneous x-ray contribution from the instrument. By carefully redesigning and remaking the specimen stage and its environment from aluminium, the instrumental contribution may be subtracted from spectra recorded on this specimens. Measurements of the apparent concentrations of Ca and P in araldite due to extraneous x-rays from other parts of the specimen, show that accurate quantitative analyses of Ca and P may be performed in unmineralized organic matrix such as osteoid, despite the proximity of fully mineralized tissue. Quantitative microprobe analyses have been performed on 1) intracellular mineral deposits, 2) osteoid, 3) mineralized matrix in trabecular bone from rachitic (R), vitamin D-treated (D) and normal (N) rats. The composition of the intracellular mineral deposits was similar in all 3 groups with mean Ca:P molar ratios = approximately 1.0. The Ca and P levels in osteoid from each group were sufficiently high that, assuming: a) that all of the Ca and P was in ionized form and b) that osteoid has a water content of 59% the calculated [Ca2+] x [HPO4(2-)] products were much greater than that required for spontaneous precipitation of mineral. In the D and N groups, the Ca and P concentrations in mineralized matrix increased with increasing distance into bone, but in the R group they remained constant. The Ca:P molar ratios of the mineralized matrix and of the serum were highest in the R group (bone: 1.85--1.87, serum: 5.76 +/- 1.05), intermediate in the D group (bone: 1.72--1.90, serum: 2.46 +/- 0.36) and lowest in the N group (bone: 1.68--1.80, serum: 1.25 +/- 0.17).