In order to describe initial events in enamel mineralization and to help characterize inorganic-organic interactions in this tissue, the earliest rod and interrod enamel in mandibular incisors from normal young adult (100 gm) rats, perfused with 100% ethylene glycol, has been studied by transmission electron microscopy, selected area electron diffraction, and high-spatial-resolution electron probe microanalysis. Diffraction and probe data were correlated precisely from the same extracellular regions of the tissue. Sites were examined progressively as a function of location a) from the most recently deposited enamel adjacent to ameloblasts toward the dentin-enamel junction and b) from the apical portion of the tooth longitudinally toward its incisal end. Electron diffraction patterns consistent with that of a poorly crystalline hydroxyapatite were generated at all locations. Diffraction characteristics changed only slightly toward that of more crystalline hydroxyapatite at different locations. Earliest apical enamel generated molar Ca/P ratios in a range of 0.99-1.46 (average 1.24 +/- 0.15). Molar Ca/P ratios of the first enamel interrod elements increased from approximately 1.24 at ameloblast-enamel boundaries to approximately 1.40 at the dentin-enamel junction, small changes corresponding to those observed in electron diffraction characteristics.

Using laser light scattering spectroscopy, we are studying age-related changes in the microstructure of lens cytoplasm. We have established in animal models that one of the earliest identifiable stages in cataract development is the presence of a phase transition in the lens cytoplasm. As a result of the phase transition, the cytoplasm separates into microvolumes that differ in their protein concentration. These microvolumes scatter light and cause the lens to become opaque. This phase separation occurs in normal lens cells at a characteristic temperature, Tcat, which varies across the lens with the cell age. As the animal becomes older, the Tcat for the nuclear cells decreases to a value well below body temperature. In X-irradiated eyes, however, Tcat increases with animal age until the phase separation occurs at or near body temperature. At this point, a well-developed nuclear cataract appears. We are now attempting to understand the biochemical basis for the differences between Tcat of normal and Tcat of X-irradiated lens cells during the aging process.

Whole enamel scrapings from teeth of embryonic calves have been separated by density gradient centrifugation in organic solvents into fractions (1.6 less than p less than 2.4 g/cm3) representing progressive stages of mineral phase maturation. Single enamel particles or their small aggregates from such fractions were examined by transmission electron microscopy, electron diffraction, and high-spatial-resolution electron probe micro-analysis. The electron optical methods demonstrated the presence of poorly crystalline hydroxyapatite as the only detectable solid phase in all fractions. Octacalcium phosphate and brushite were not identified in the fractions. Changes in electron diffraction patterns were indicative of a progressive increase in apatite crystallinity with enamel maturation. Molar Ca/P ratios were found to range from 1.48 to 1.70, with a higher mean value obtained for lower-density fractions (p less than 2.0 g/cm3). Lower-density fractions contained some particles with high ratios (approximately equal to 2.0-4.0) and a non-uniform distribution of Ca and P, as revealed by electron probe mapping. These characteristics are suggested as possibly being related to carbonate phases in early enamel.

The outer cortical cells in the calf lens remain transparent under conditions that produce opacity in central nuclear cells. The nuclear cells opacify by a mechanism of cellular restructuring that is associated with a cytoplasmic phase separation while cortical cells do not opacify by this mechanism. In this study the differences in elemental composition of nuclear and cortical cells were analyzed using X-ray emission spectroscopy (XES) of tissue that was prepared for scanning electron microscopy. It was necessary to develop special methods of fixation and dehydration to prevent significant distortion of lens tissue and minimize solubilization and redistribution of elements during the histological processing of the tissue. We calibrated the microprobe for the quantitative analysis using gelatin standards which contained known concentrations of sulfur, potassium, phosphorus, chlorine, and cesium. The standard curves were used to determine proportionality constants, which related the intensity of X-ray emission to the molar concentration of each element, and to determine the minimum detectable levels of each element. An important finding is that the intensity of the X-ray emission is dependent on sample density only at low protein concentration. At the high protein concentrations that exist in lens, the intensity is not affected by sample density.(ABSTRACT TRUNCATED AT 250 WORDS)