An energy dispersive x-ray analysis study of elemental loss from globoid crystals in protein bodies as a result of osmium tetroxide fixation

Abstract

This study was undertaken to discover what elemental losses, if any, were occurring from globoid crystals in seed protein bodies during glutaraldehyde – osmium tetroxide fixation. Unfixed cotyledon and radicle tissue of Cucurbita maxima seed, or tissue after glutaral–dehyde–OsO4 treatment, was quick frozen in liquid N2, ground with a cold mortar and pestle, and low-temperature freeze-dried. Globoid crystals in the freeze-dried powder were subjected to energy dispersive x-ray (EDX) analysis. OsO4 fixation resulted in a major loss of P, Mg, and K from cotyledon globoid crystals and a major loss of P, Mg, K, and Ca from radicle globoid crystals. Despite the loss of elements, the OsO4-fixed globoid crystals were still electron dense. When globoid crystals from glutaraldehyde-fixed, dehydrated, and embedded cotyledon tissue were compared with globoid crystals from glutaraldehyde–OsO4-fixed, dehydrated, and embedded tissue, some extraction was found. The degree of extraction varied from complete loss of P, K, and Mg to loss of K only.Effects of glutaraldehyde–OsO4 fixation upon elemental composition of globoid crystals in several other species was also determined. Brazil nut (Bertholletia excelsa) radicle tissue or cotyledon tissue from walnut (Juglans regia), hazelnut (Corylus avellana), sunflower (Helianthus annuus), golden everlasting daisy (Helichrysum bracteatum), cashew (Anacardium occidentale), pistachio (Pistacia vera), and the Western Australian red-capped gum (Eucalyptus erythrocorys) were fixed either in glutaraldehyde or in glutaraldehyde–OsO4. In these species, EDX analysis of globoid crystal sections showed that OsO4 fixation results in major loss of Mg, K, and Ca. Generally, phosphorus levels were reduced from control values as well. When carrying out EDX analysis studies of globoid crystals, we recommend (1) avoiding any use of OsO4, (2) keeping all fixation, washing, and dehydration times as short as possible, and (3) checking all observations with freeze-dried powders.