Recent Developments in Methacrylate Embedding

The increasing use of thin sections of methacrylate-embedded specimens (I) as a means for the study of the fine structure of cells and tissues with the electron microscope has accentuated the problem of the elimination of preparation artifacts. The high resolution of this relatively new instrument has made it necessary to think in terms of the preservation of structures with dimensions well below the limit of resolution of the optical microscope. One approach to this problem has been provided by a technique, developed by Borysko and Sapranauskas, that permits the continuous optical examination of cells, grown in tissue culture, through all phases of the methacrylate embedding process, from the living state to the final embedded condition (2). Although this technique is limited to the optical level of resolution, it can be reasonably assumed that submicroscopic artifacts would be minimal when the appearance of the cells in the embedded condition is optically identical with their living aspect as seen just prior to fixation. Originally, Borysko and Sapranauskas did not detect any alterations in the appearance of the cells throughout the entire methacrylate embedding process. However, subsequent experience revealed that cells in many embeddings were obviously badly distorted, and a re-evaluation of the method was undertaken. I t was found that, though considerable shrinkage of spherical cells occurred during dehydration with ethanol, the most serious distortions frequently occurred during the polymerization of the embedding matrix. In this report, a study of the characteristics of polymerization damage is presented and a method for the production of relatively undamaged embedded specimens is described. A hypothesis for the mechanism of polymerization damage is proposed, based on the data obtained and the physical changes that are known to take place in methacrylate during polymerization.