Freeze-substitution and Epon embedment were quantitatively evaluated for their effectiveness in retaining water-soluble metabolites in plant tissues. Roughly 99% of the 80% (v/v) ethanol-extractable radioactivity in photosynthetically labeled soybean leaf discs and in petiole fragments containing translocated "C was retained during freeze-substitution in acetone or propylene oxide and embedment in Epon. Substantially more activity was lost from "C-sucrose-infiltrated pith blocks, but most or all of this loss came from the block surface. The procedure was effective for a sucrose concentration as low as 0.004%. Sections floated on water retained most of their UC sucrose, and high resolution autoradiographs could easily be prepared without resorting to dry procedures. Embedded "Csucrose was apparently chemically unreactive, since there was no loss of radioactivity when sections were stained with the periodic acid-Schiff reagent, nor did the embedded sucrose show staining.Histological procedures for the localization of water-soluble compounds in the phloem have followed in large part the experimental approach outlined by Branton and Jacobson (2). This consists essentially of freeze-drying the tissue and, with a recent exception, embedment in paraffin, followed by "dry autoradiography." Fritz and Eschrich (7) have recently vastly improved on this approach by treating freeze-dried tissue with silicone and then embedding in Epon, rather than in paraffin. However, in all cases where sufficient resolution was attained, an irregular distribution of silver grains over the sieve tube lumen was observed (1,7,9,12,15,16).During earlier work with freeze-substitution, it was observed that at least some water-soluble carbohydrates were retained by freeze-substitution and Epon embedment but were lost during aqueous aldehyde fixation (6). While developing this procedure for the embedment of sugars for microscopy, it was also found that the water-soluble contents of cells in freeze-dried preparations were very susceptible to shrinkage (5). This shrinkage might explain the apparent uneven distribution of radioactive water-soluble compounds in the sieve tubes of freeze-dried phloem. With care, this artifact could be virtually eliminated from freeze-substituted tissue, but not from 'This work was supported in part by Grant GB-14719 from the National Science Foundation. freeze-dried tissue. Freeze-substitution, then, seemed to offer the double advantage of providing a much simpler alternative to freeze-drying as well as a more accurate cytological picture of the tissue's water-soluble constituents. Furthermore, embedment of the tissue in Epon greatly improves both the autoradiographic resolution (thinner sections) and the ease of preparing autoradiographs (7, 13). Epon also physically protects the cell structure from the adverse effects of water, thus allowing meaningful cytological observations of the same material.MATERIALS AND METHODS Plant Tissues. Tissue containing "C-labeled metabolites was provided by photosynthetic i...