128 MACROMOLECULAR COMPLEXES 



directly in electron micrographs of osmium-fixed thin sections (e.g., 

 Figs. 9, 10). Shrinkage effects introduced by the preparation tech- 

 niques account for the difference of 20 to 40 A between the two 

 values; and from a detailed analysis carried out on the same speci- 

 men at each stage of the process, quantitative data were obtained 

 on the dehydration and embedding artifacts ( Fernandez-Moran 

 and Finean, 1957). 



Against this background, which provides a reliable and detailed 

 correlation between the organization of lamellar structures in the 

 fresh myelin sheath and the corresponding electron microscope 

 images, it is evident that mvelin constitutes one of the best systems 

 for a step-by-step validation of the results of the new preparation 

 methods. In the following, we shall first consider the main artifact 

 sources derived from dehydration and embedding at room tempera- 

 ture, before dealing with the ice-crystal artifacts. 



Effects of Dehydration and Embedding. When the sciatic 

 nerve from a living frog or guinea pig is rapidly frozen in Freon 

 22-liquid nitrogen at approximately —160° C, subjected to freeze- 

 substitution in a 1 per cent osmium tetroxide-acetone mixture at 

 —75° C according to the procedure of Feder and Sidman (1958), 

 and then embedded in methacrylate or araldite at 40° to 55° C for 

 ultrathin sectioning, certain modifications are regularly observed. 

 Although the myelin sheaths of a few small fibers appear to be well 

 preserved, most nerve fibers show extensive vacuolization of the 

 sheath, gross distention, and other rearrangements leading to partial 

 or complete disruption of the layers. The poor preservation of fine 

 structure revealed by the high resolving power of the electron micro- 

 scope stands in marked contrast to the well-preserved appearance 

 under light microscope examination of a thicker section taken from 

 the same block. The obliteration of fine structure, although caused 

 in part by ice-crystal artifacts, is largely due to the extraction of the 

 hpoprotein components of the layers during treatment with the 

 methacrylate monomer, which acts as a lipid-extracting agent, par- 

 ticularly at embedding temperatures of 40° to 55° C. Bv contrast, 

 remarkable improvement in the preservation of the m\elin sheath 

 structure is immediately noted when impregnation with the mono- 

 mer and subsequent photopolymerization are carried out at tem- 

 peratures of —80° to —30° C. These observations suggest that os- 

 mium tetroxide does not fix the lipoprotein constituents as effectively 

 at low temperatures as at 0° C. Low-temperature embedding is 



