Three-dimensional visualization of calcification during scale regeneration in goldfish

We used scanning electron microscopy to visualize the regeneration of goldfish scales on day 3 in vivo. Several vesicle-like structures of 100-700 nm diameter flowed onto the fibrous sheets in groups of spindle-shaped bodies arranged in the same direction. Transmission electron microscopy revealed that these structures were encircled by the lipid bilayer membrane. In addition, some had a small mass of high electron density. Scanning electron microscopic observations of specimens treated with bleach revealed particles of almost the same size as the observed electron-dense mass scattered between fibers, with a thickness of approximately 50 nm on day 3 of scale regeneration. The diameter of these particles increased by 5 times on day 14, sticking closely to the fibers. Furthermore, elemental analysis using electron probe microscopy showed that the particles were composed of calcium and phosphorous. These results confirmed that the spindle-shaped bodies and vesicle-like structures were osteoblasts and matrix vesicles, respectively.