A comparison of subcellular element concentrations in frozen-dried, plastic-embedded, dry-cut sections and frozen-dried cryosections.
Biological X-ray microanalysis of diffusible elements within cellular and subcellular compartments requires preparation methods to retain electrolytes in the compartments they occupied in vivo. X-ray microanalysis of frozen-dried, plastic-embedded samples has been used to quantitate electrolytes at the cellular level. We have compared the subcellular elemental distribution in dry cut sections from such samples with that in ultrathin frozen-dried cryosections. Rat pancreases were quench-frozen onto a helium-vapor-cooled copper block. Cryosections were cut at 130-150 K, transferred using a Gatan cold stage, frozen-dried in the column and analysed at 190 K. Tissue fragments were frozen-dried at 190 K, and cut on a dry knife at 293 K. Both samples provided images permitting unambiguous identification of all major compartments except the Golgi complex. Intracellular potassium-to-sodium ratios obtained on frozen-dried plastic-embedded sections were lower than for cryosections (e.g. 1.77 in basal cytoplasm in plastic sections as compared to 4.34 for cryosections) and varied with the pre-embedding procedure (e.g. 1.77 in formaldehyde-fixed as compared to 2.87 in osmium-fixed plastic sections). Potassium gradients between adjacent organelles were large in cryosections and insignificant in plastic-embedded material. Higher cytoplasmic phosphorus, potassium and sulfur concentrations were observed in cryosections. Therefore, a redistribution of electrolytes and covalently bound elements occurred subcellularly in the plastic sections. Calcium was quantifiable in most organelles in cryosections but the plastic lowered sensitivity too much to permit routine calcium quantification. We conclude that in our hands frozen-dried, plastic-embedded samples were compromised and provided lower sensitivity than cryosections.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002118:Calcium / Q000032:analysis; D004577:Electron Probe Microanalysis / Q000379:methods; D004602:Elements; D004853:Epoxy Resins; D005612:Freeze Drying / Q000379:methods; D005629:Frozen Sections / Q000295:instrumentation / Q000379:methods*; D008854:Microscopy, Electron; D008867:Microtomy / Q000379:methods*; D010179:Pancreas / Q000032:analysis / Q000648:ultrastructure*; D011188:Potassium / Q000032:analysis; D051381:Rats; D012964:Sodium / Q000032:analysis; D013347:Subcellular Fractions / Q000032:analysis / Q000648:ultrastructure; D013455:Sulfur / Q000032:analysis
D004602:Elements; D004853:Epoxy Resins; C004875:EPON; D013455:Sulfur; D012964:Sodium; D011188:Potassium; D002118:Calcium