LAMELLAR SYSTEMS 137 



its potential value for electron microscopy. Glycerinated nerve 

 could not be examined directly with the techniques which were 

 available at the time, because glycerol is not miscible in methacry- 

 late, and the required treatment with alcohol followed by embed- 

 ding at 45° C would have destroyed the unfixed lipoprotein struc- 

 tures. 



However, with the development of the cryofixation techniques, 

 the glycerol permeating the tissues could be substituted at ade- 

 quately low temperatures with alcohol or, preferably, methyl cello- 

 solve, and the tissues impregnated with the acrylic monomers and 

 finally photopolymerized under favorable conditions which would 

 minimize extraction and embedding artifacts. The highly encourag- 

 ing results obtained with this technique have established it during 

 the past year as the method of choice when dealing with larger 

 tissue blocks or even whole small organs. Since equilibration with 

 buffered glycerol solutions of increasing concentration can be per- 

 formed gradually at temperatures of —5° to —35° C, the usual de- 

 hydration and extraction effects are markedly reduced. Once a 

 glycerol concentration of 40 to 60 per cent has been attained, the 

 tissue can be cooled to — 75° C or lower, and the entire preparation 

 cycle completed at these "safe" temperatures. When the embedded 

 tissue is brought up to room temperature for sectioning, the re- 

 markable preservation of the original configuration and translucent 

 natural colors stands in marked contrast to the opaque and artificial 

 appearance of other preparations. Electron microscopy confirms 

 that a high degree of structural integritv has been achieved, and it 

 is instructive to see a cross-section through a whole "unfixed" retina 

 perfectly intact, and free from ice-crystal artifact. In glycerinated 

 low-temperature preparations, the lamellar systems and the nuclear 

 structures are better preserved than the loose cytoplasmic matrix; 

 the remaining artifacts may in fact be reduced when the equilib- 

 rium with glycerol can be refined or supplemented with a more 

 physiological protective agent. Examination of thin sections of 

 glycerinated nerve fibers (Figs. 7, 9) which have been stained with 

 osmium discloses an expanded myelin layer spacing of the order of 

 150 to 160 A, with clear-cut indications of a granular fine structure 

 of the intermediate and dense layers. In several areas this particu- 

 late fine structure exhibits a certain degree of regularity in the 

 radial direction. A satisfactory correlation of the x-ray diffraction 

 patterns with the electron micrographs must await further studies; 



