LAMELLAR SYSTEMS 129 



therefore essential for all freeze-substitiition procedures, and has 

 proved to be the most important single factor, actually making 

 possible the systematic application of low-temperature preparation 

 techniques to the stud\' of the labile lamellar systems. 



Earlier x-ray and electron microscope studies of the standard 

 preparative procedures (Fernandez-Moran and Finean, 1957) had 

 already disclosed complex modifications, including, first, a shrink- 

 age of the myehn layer spacing during osmium fixation and alcohol 

 dehvdration, then a compensating expansion during methacrvlate 

 embedding. The significant extraction of material known to occur 

 during fixation and dehydration (Miiller, 1957) was therefore being 

 partially masked by the methacrylate embedding. Although such a 

 detailed analysis of the modifications introduced during fixation, 

 dehydration, and embedding at low temperatures remains to be 

 carried out, possible mechanisms which might be operative under 

 these conditions are already suggested. Since the macromolecular 

 matrix of the myelin lamellae, frozen and stiff, is thus maintained 

 essentially immobilized during impregnation with the monomer and 

 subsequent photopolymerization at low temperatures, the lipid ex- 

 traction effects are not only considerably reduced, but the whole 

 system is permanently "set" or fixed in its natural position by the 

 polymerizing embedding medium before it is brought up to room 

 temperature. In this way, major rearrangements of fine structure 

 which might otherwise take place during the process of thawing 

 are largely avoided; and investigation of ice-crvstal artifacts can 

 now be profitably undertaken. 



Effects of Ice-Crystal Formation. The comprehensive x-ray 

 diffraction studies carried out by Finean (1958) have shown that 

 definite structural modifications occur after rapid freezing and thaw- 

 ing of nerve. In peripheral nerve, this usually results in a complete 

 halving of the radial repeating unit and partial disorganization of 

 the layers. The corresponding electron micrographs demonstrated 

 characteristic changes ranging from an increased general structural 

 breakdown of the layers to a pronounced accentuation and broaden- 

 ing of the intermediate line, which could be correlated with the 

 halving of the radial repeat and abolishing of the difference factor 

 (Fernandez-Moran and Finean, 1957). These changes are brought 

 about primarily by transient formation of submicroscopic ice crys- 

 tals and associated freezing effects in the compact myelin sheath. 

 They can therefore serve as a reference for evaluation of the results 



