Artifacts in the Embedment of Water-soluble Compounds for Light Microscopy

The relative effectiveness of freeze-drying and freeze-substitution in accurately preserving the distribution of water-soluble compounds in plants tissues was evaluated. Specimens with a known distribution of water-soluble materials (sucrose-infiltrated pith blocks and red beet tissue) were freeze-dried or freeze-substituted and embedded in paraffin, Epon, or methacrylate for microscopic examination. Dried water-soluble compounds were extremely sensitive to only traces of water, which caused severe shrinkage of the vacuolar contents. In spite of care taken to maintain dry conditions, freeze-drying was always accompanied by marked shrinkage of the water-soluble compounds away from the cell walls. Freeze-substitution was successful only if the solvents and embedding media were thoroughly dried and if infiltration was done in a dry box. Other artifacts were also noted, although they were less severe. Artifacts comparable to those seen in pith blocks and in red beet tissue were observed in the contents of pumpkin sieve tubes.