Freeze-Substitution Additives for Sodium and Calcium Retention in Cells Studied by X-Ray Analytical Electron Microscopy

In salinity studies on the cultivated tomato, Lycopersicon esculentum Mill, seedlings were grown in solution cultures containing 0, 40, and 120 meq/1 NaCl for observation by X-ray analytical electron microscopy Freeze-substitution systems based on diethyl ether and containing additives were tested for their effect on the retention of sodium in young leaf tissue. The additives were picrolonic acid, 2,6-dinitrophenol, alizarin, benzamide, and oxalic acid. Cyanuric chloride was also tested as a nonmetal-containing fixative in the freeze-substitution fluid Only benzamide resulted in the retention of significant levels of sodium in the tissue, and it was also effective for calcium Peak-to-background ratios as high as 6.2 were achieved for sodium and 48 0 for calcium on an X-ray dispersive spectrophotometer of EMMA-4. Distinct differences in the relative amounts of these elements were recorded at different ultrastructural sites in the cells, possibly reflecting their distribution in living tissue The processing involved quenching the fresh tissue at -160 C in 2-methyl butane (isopentane) cooled by liquid nitrogen and then transferring the tissue to diethyl ether dehydrated with Al2O3 and saturated with the freeze-substitution additives Freeze substitution proceeded for 8 days at about -80 C under dry ice in a -20 C freezer Two changes of the fluid were made The tissue was then warmed to -20 C and infiltrated with vinyl cyclohexene dioxide. This was followed by infiltration with Spurr's low-viscosity epoxy resin at ambient laboratory temperatures and then polymerization Noncontrasted sections 1,500 Å thick for observation and analyses by EMMA-4 were cut dry at -50 C with a Reichert cryomicrotome or wet with a Cambridge ultramicrotome at ambient laboratory temperatures Thin sections observed by conventional electron microscopy exhibited positive contrast through grid staining with uranyl acetate and lead citrate and excellent preservation of the general structure and of the major organelles Numerous minor artifacts were attributed largely to ice formation