An excursion through the ultrastructural world of quick-frozen pancreatic islets



In this article we have presented a philosophical and historical perspective on quick freezing, freeze-drying, freeze-substitution, and immunocytochemical localization of pancreatic islet hormones. A compilation of our findings indicates that (1) quick-freezing does not produce any gross distortion of islet tissue; (2) the amount of usable islet tissue for ultrastructural analysis is approximately 13 μm deep from the frozen edge; (3) three different cell types can be identified in quick-frozen tissue based on general morphological characteristics; (4) freeze-substitution with tetrahydrofuran produces a unique ultrastructural appearance in which ribosomes are particularly striking; (5) with the use of protein A-gold, insulin and glucagon can be localized immunocytochemically on silver-gray (50-nm-thick) sections treated with 1% ovalbumin at room temperature overnight; (6) secretory granules of quick-frozen alpha and beta cells may exist in either a swollen or condensed state; (7) swollen beta cell secretory granules contain a filamentous material that demonstrates immunogold labeling for insulin; (8) insulin and glucagon can be localized within the cisternae of endoplasmic reticulum; (9) our methods provide not only discrete immunocytochemical localization of hormone, but also well-preserved cellular compartments; (10) energy electron loss spectroscopy (EELS) has shown that quantifiable nitrogen maps can be used as an index of hormone packaging in secretory granules; and (11) the sectioning properties of secretory granules at the ultramicrotome change when islet tissue is unosmicated and sectioned on glycerol.