Preparation of thin cryo-sections for electron probe analysis of calcifying cartilage.

Abstract

Conventional methods of fixation, dehydration, embedding and wet-sectioning can produce artefacts in the chemical composition of mineralizing tissues. Cryoultramicrotomy was adopted for a more reliable approach to electron probe analysis of initial apatite formation in calcifying cartilage. Fresh rabbit epiphyseal cartilage was mounted on silver pins, frozen by immersion in liquid nitrogen, and sectioned with the specimen temperature at 133 K and the knife temperature at 273 K. Dry cryo-sections (30-70 nm in thickness) were manipulated on to coated grids and examined the same day. These cryo-sections showed good morphological and cytoplasmic detail, with large areas relatively free of ice-crystal damage. Sections stained either with osmium vapour or negatively stained with silicotungstic acid showed areas with well-preserved mitochondria with granules and endoplasmic reticulum. Unstained sections also showed dense granules (50-120 nm in diameter) in the mitochondria of chondrocytes and preliminary electron probe analysis of these has indicated a Ca/P mass ratio of approximately 1.14. In the longitudinal septa, about 2 micrometer away from the chondrocytes, matrix-vesicle-like particles could be seen with crystal needles inside them. Micro-analysis of two of these gave a Ca/P mass ratio of 1.73 and 2.68. Cryo-ultramicrotomy appears to confirm a number of conclusions derived from conventional ultrastructural study of growth cartilage and suggests for the first time how amorphous calcium phosphate and crystalline apatite can be shown to exist in different organelles in the same cryo-section of the tissue.