Freeze‐substitution of scleractinian coral for confocal scanning laser microscopy and X‐ray microanalysis

SUMMARY Freeze‐substitution in combination with confocal scanning laser microscopy provides a unique means by which the microstructure of undecalcified, and chemically untreated, polyps of scleractinian corals can be examined free of preparation artefacts. Mucocytes and intercellular spaces are particularly well preserved and the relationships of cell layers to each other and to the skeleton are undisturbed. Freeze‐substitution also permits X‐ray microanalysis of bulk samples and thin sections, a procedure which has hitherto been impossible to carry out on corals except on fixed tissue samples. Analyses indicated a high retention of Na+ and Cl− in spaces thought to be filled with fluid similar in composition to sea water. This increases confidence in freeze‐substitution as a means of retaining diffusible ions. Zooxanthellae contained metal/Ca ratios within the range of those previously reported for extracted zooxanthellae. It is shown that the mucocytes contain very high concentrations of S, K, Ca and Sr which are specifically localized in mucous granules. The concentrations differ between, and are characteristic of, different epithelial cell layers. Remarkably good correspondence was obtained between the two entirely different X‐ray analytical methods. This is the first time such a comparision between methods has been made. It is suggested that the uptake of transepithelially transported Ca2+ and Sr2+ by mucocytes may be a means of regulating the deposition of these ions in the skeleton.