Demonstration of calcium ion distribution in calcifying cells - X-ray microanalysis and electron spectroscopic imaging after fixation with NHA-containing fixative and microwave irradiation

We have seen reporting on the calcium ion distribution in cells and tissues with X-ray microanalysis (EDX) since 1978 [9] and with electron energy-loss spectroscopy (ESS) imaging since 1983 [10, 17], under various fixing conditions [12-16, 18-24]. Almost ten years ago, we tried a fixative containing NHA (N,N-naphthaloylhydroxamine) as a chemical precipitant for Ca2+ ions under microwaved conditions [12, 19]. However, the data did not seem to be as sensitive or useful, under conventional electron microscopy and computerized X-ray microanalysis, compared to the two-step replacement method combined with microwave fixation using potassium (K) oxalate followed by potassium (K) antimonate, for the detection of Ca2+ ions [12, 19-21]. Recently, however, we used ESS to analyze same tissue sections which were fixed with NHA-containing fixative with microwave fixation ten years previously. We discovered with ESS analysis that NHA is a sensitive and ideal chemical reagent for calcium ion detection in a cell giving the same view as that of frozen dried or freeze-substituted tissue sections. Calcium was clearly seen and widely distributed in developing chondroblast endoplasmic ultrastructures: ribosomes, cell and endoplasmic membranes, mitochondria, Golgi, and nucleus. However,free mesenchymal cells which were not involved in calcifying functions had only a very small amount of calcium. Calcium was widely distributed in the lacunae of the surrounding space of the chondroblasts and inmature matrix, but the exoplasm of the chondroblasts was almost calcium-free. Other elemental images in the young chondrocytes or osteoblasts, i.e., P.L, S.L, O.K, and N.K, were seen clearly superimposed on the cell ultrastructures, i.e., the cell membranes, ribosomes, endoplasmic reticula, mitochondria, Golgi, and nucleus [24].