Methods of Preparation of Fossil Bone Samples for Light and Transmission Electron Microscopy

Abstract

The methods described make possible the preparation of fossil bone samples for light and transmission electron microscopy. To obtain good images with ground sections of bone, both the grinding process and classical bone staining techniques were modified, the latter by staining already made ground sections. Ground sections of suitable thickness were etched with a 4:1 mixture of 5% HNO₂ and 2% OsO₄ at a lowered temperature, thereby obtaining isolated osteocytes with a complete network of processes. Using the same method with thicker ground sections, the contents of vascular canals were isolated. The contents retained spatial relations permitting complete reconstruction of the vascular system of the bone in question. The isolated osteocytes, the vascular canal contents, and the isolated remnants of the intercellular organic part of this bone, subsequently embedded in Epon 812 and in methacrylate, made possible sections suitable for detailed analysis of the image of the surfaces of these elements. Polished surfaces of the fossil bone were replicated by the so-called clearing replicas technique. This was followed by replication performed with the modified combined replicas technique adapted to fossil bone. The latter facilitated analysis of the ultrastructure of the surfaces of the vascular canals, isolated osteocytes and their processes, and analysis of the immediate proximity of these elements, since the technique preserves the mutual spatial relations intact.