LAMELLAR SYSTEMS 131 



scopic ice crystals within the layered structure. The images ob- 

 tained often resemble the structural modifications in sciatic nerve 

 described earlier (Fernandez-Moran and Finean, 1957) after rapid 

 freezing and thawing, standard osmium fixation, and methacrvlate 

 embedding. When fresh nerves were rapidly frozen in liquid helium 

 I (—269° C) and then thawed, a characteristic multiple splitting of 

 the dense and intermediate lines was noted (Fernandez-Moran and 

 Finean, 1957), in contrast to the more homogeneous appearance 

 and broadening of the layers encountered after rapid freezing in 

 liquid nitrogen. In cryofixation preparations, there appears to be a 

 more difl^erentiated interaction of these freezing effects with the 

 ordered lamellar substrate, leading to varying degrees of enhanced 

 structural definition. Thus, the better preservation of tissue fine- 

 structure generally observed in specimens frozen with liquid helium 

 II, as compared with control preparations cooled with other refrig- 

 erants, might be related to the initial formation of finer ice crystals, 

 and to other as-yet-unknown auxiliary factors which could con- 

 tribute to the stabilization of the macromolecular matrix. However, 

 elucidation of these essential problems must await further analysis 

 of myelin ultrastructure performed directly at liquid helium tem- 

 peratures by x-ray diffraction techniques, and pursued systemat- 

 ically, in combination with electron microscopy, throughout the 

 successive preparative manipulations. 



Osmium Cryofixation. When osmium cryofixation is carried out 

 on fresh frog sciatic nerve under conditions minimizing the de- 

 scribed artifacts, the myelin sheath in most of the outer fibers is 

 unusually well preserved. As shown in Fig. 6, the highly regular 

 aiTangement of the concentric layers is clearly visible over extensive 

 areas of the sheath, giying an over-all impression of precise align- 

 ment, which derives from immobilization of the straight fiber bun- 

 dles in their stiffened state at low temperatures. The fine structures 

 revealed are essentially similar to those detected in the best stand- 

 ard osmium-fixed preparations. The "unfixed" thin sections stain 

 intensely with a wide variety of reagents, including uranyl acetate, 

 phosphotungstic acid, lead hydroxide, and iodine and bromine com- 

 pounds, disclosing a similar type of laminated structure in the 

 sheath, with characteristic variations which are being investigated in 

 greater detail. Evidence of fine structure within the layers or split- 

 ting of the dense and intermediate lines (Figs. 7-11), in addition to 

 a more compact and dense appearance of the interspace, were con- 



