Electron probe analysis of cryosections of epiphyseal cartilage

Conventional methods for preparing specimens for analysis of the chemical composition of mineralizing tissues at the electron microscopic level can produce artefacts during fixation, dehydration, embedding and wet-sectioning. Cryoultramicrotomy provides a more reliable approach to electron probe analysis of initial apatite formation in calcifying cartilage. Two procedures are described (a) for a micro-analytical approach and (b) for a morphological study. By using a negative staining procedure (b) cytoplasmic details in terms of subcellular organelles and extracellular, membrane-bound, matrix vesicles could easily be identified in relatively large sections of epiphyseal cartilage. Unfixed, dry, unstained cryosections obtained by procedure (a) showed dense mineral granules (50–120 nm in diameter) in the mitochondria of chondrocytes and electron probe analysis of these gave a mean Ca/P mass ratio of 1.20. Adjacent cytoplasmic areas showed negligible levels of calcium. In the longitudinal septa matrix, vesicle-like particles could be identified with characteristic crystal needle clusters. Microanalysis of these structures showed them to have a mean Ca/P mass ratio of 2.15. Non-vesicular matrix appeared to be very low in calcium and phosphorus. The combination of cryoultramicrotomy and electron probe analysis of different areas in the same cryosection has provided some information on the coexistence of amorphous calcium phosphate in the mitochondria and of crystalline apatite in matrix vesicles a few micrometers away. These findings may thus explain the sequence of hydroxyapatite formation in epiphyseal cartilage.