Use of backscattered electron imaging on developed radioautographic emulsions: Application to viewing rat incisor enamel maturation pattern following ⁴⁵calcium injection

Abstract

Backscattered electron imaging (BED can be used to obtain compositional contrast in biological structures because it detects differences in concentration of elements with different atomic number. Rat incisor enamel shows a banded pattern in the maturation zone when radioautography is used to reveal the location of injected 45Ca at the enamel surface. The bands of developed silver grains in the photographic emulsion that coats the enamel surface are ideal for atomic number contrast. The purpose of this study was to use BE1 to analyze the radioautographic pattern with SEM resolution. Onemonth-old rats were injected with 45Ca and sacrificed at early (1 min, 5 min, 30 min), intermediate (4 h, 8 h), and late (1 day, 4 days) time intervals after injection. Whole incisors were dissected, the enamel organs were removed, and the enamel surface was coated with photographic emulsion and processed for radioautography. These radioautographed teeth were examined with a JEOL JSM-840 SEM equipped with a JEOL backscatter annular-type detector. At the early time intervals, light microscopic examination showed five or six broad black bands running obliquely across the teeth. These were separated by narrow white bands of unlabeled enamel. BE1 resolved the presence of a delicate subbanding pattern within each dark band. At the intermediate time intervals, although the incisal bands persisted, only a diffusely blackened apical area was seen by light microscopy. BE1 resolved bands in this apical region, but these showed no subbanding pattern. At the late time intervals, both light microscopy and BE1 showed no banding, and the enamel was uniformly labeled. The significantly improved resolution obtained with BE1 on surface radioautographs has revealed a previously undetected substructure in the 45Ca-labeled banding pattern seen in enamel maturation.

Effects of vinblastine on calcium distribution pattern and Ca2+,Mg(2+)-adenosine triphosphatase in rat incisor maturation ameloblasts.

“…Considerable evidence supports the view that RA exert more influence over mineral uptake in adjacent enamel than SA. This has been demonstrated autoradiographically (McKee et al, 1987;Takano et al, 1982a,b;Reith and Boyde, 1981) and histochemically (Takano et al, 1988). The regions of enamel showing the greatest uptake of 45Ca, which is believed to be incorporated into hydroxyapatite, are adjacent to RA (Takano et al, 1982b;Reith and Boyde, 1981).…”

Cellular and Chemical Events During Enamel Maturation

“…It has been known for many years that both 45Ca and 32P04 appear very rapidly (from seconds to minutes) within the mineral phase of developing enamel, as in other hard tissues, after systemic injections are given, especially those done intravenously (Munhoz and Leblond, 1974;Robinson et al, 1974;Reith and Boyde, 1981;Reith et al, 1984;McKee et al, 1987;Uchida et al, 1987;Hanawa et al, 1990;Shimizu, 1990, 1997). In the case of enamel, the entry point for these ions is from blood vessels on the surface covered by enamel organ cells (Bawden, 1989;Eisenmann et al, 1989;Takano, 1995).…”

Banding Patterns in rat incisor enamel stained by histochemical complexing methods for calcium

“…These methods demonstrate the anion, such as phosphate and carbonate, rather than the cation. Of particular interest during enamel maturation is the pathway(s1 taken by the predominant cation, calcium, since this mineral precursor differentially enters maturing enamel in a banded pattern Takano et al, 1982a;Reith et al, 1984;McKee and Warshawsky, 1986a;McKee et al, 1987). Since numerous colorimetric and photometric methods have been developed for the detection of soluble and insoluble metal ions and ion complexes, several of these were chosen to visualize calcium at the surface of rat incisor enamel.…”