LAMELLAR SYSTEMS 127 



In general, satisfactor>' preservation of the integrity and fine struc- 

 ture of lamellar systems could be achieved by rapid freezing of thin, 

 fresh specimens or of glycerol-treated larger tissue samples, prefer- 

 ably with liquid helium II, followed by controlled freeze-substitution 

 at temperatures of —130° to —80° C, and low-temperature poly- 

 merization. However, thin sections of this "unfixed" material 

 showed such lability and low contrast when examined directly in 

 the electron microscope that staining with heavy-metal salts was 

 found to be necessary, except in the case of nuclear components 

 which exhibit sufficient contrast and structural detail even in un- 

 stained preparations. In order to draw a more direct comparison 

 of the new procedures with standard electron microscopy tech- 

 niques, the findings presented will be confined mainly to those ob- 

 tained with osmium tetroxide staining, either introduced during 

 freeze-substitution or applied in vapor form to the "unfixed" ultra- 

 thin sections. 



However, in addition to the well-preserved regions, there are still 

 many areas in the specimens which exhibit ice-crystal artifacts, 

 artificial rearrangements, and other complex modifications intro- 

 duced by the new low-temperature methods. Evaluation of these 

 preparation artifacts has not only served to emphasize the limita- 

 tions and pitfalls of the new techniques, but also provided valuable 

 indications for improved preservation of the peculiarly labile hy- 

 drated state of the lipoprotein components in lamellar systems. 



Fine Structure of the Nerve Myelin Sheath. Selection of the 

 nerve myelin sheath as a standard reference system for comparative 

 studies of lamellar fine structure was clearly indicated in view of the 

 extensive data already available on normal and modified myelin 

 (Fernandez-Moran, 1957, 1959b; Finean, 1958; Schmitt, 1959; 

 Schmitt et ah, 1935). The correlative investigation of the prepara- 

 tion procedures and experimental modifications, achieved by sys- 

 tematic application of x-ray diffraction methods and electron micros- 

 copy (Fernandez-Moran, 1959b; Fernandez-Moran and Finean, 

 1957; Finean, 1958; Schmitt et al, 1935), has been of particular 

 value in defining the basic structural parameters of the fresh myelin 

 sheath. Thus, the fundamental radial repeating unit of 170 to 174 A 

 shown in the low-angle x-ray diffraction pattern recorded from fresh 

 amphibian nerve (Fig. 8) corresponds to the layer spacing of the 

 myelin sheath with an average "period of 130 to 140 A, as seen 



