Preparation methods for quantitative electron probe X-ray microanalysis of rat exocrine pancreas: a review

Pancreatic acinar cells are thought to secrete a fluid containing digestive enzymes and electrolytes and use e.g. calcium as a second messenger upon stimulation. Together with their pronounced morphological polarity, they provide a model system to study the effect of different preparation methods for quantitative biological electron probe X-ray microanalysis (EPXMA) of ultrathin sections. Several preparation methods i.e., freeze-drying and plastic-embedding, freeze-substitution (2 days) and freeze-drying of ultrathin cryosections have been applied to examine the retention of sodium, magnesium, phosphorus, sulfur, (chlorine), potassium and calcium in subcellular compartments (basal cytoplasm, apical cytoplasm, mitochondria and zymogen granules). In freeze-substituted samples the phosphorus, potassium and sulfur concentrations were 2-3 times lower in all compartments compared to freeze-dried, plastic-embedded samples. Intracellular potassium-to-sodium ratios obtained on frozen substituted and frozen-dried, plastic-embedded samples were considerably lower than for cryosections. Element gradients between adjacent organelles were large in frozen-dried cryosections, smaller in frozen-dried plastic- embedded samples and insignificant in frozen-substituted samples.