116 MACROMOLECULAR COMPLEXES 



stituents of cellular organization in tissues, including the predomi- 

 nant water component, the transient intermediates with unpaired 

 electron-spin (which participate in enzymatic reactions and meta- 

 bolic electron transfer), and other unstable chemical species gen- 

 erally referred to as free radicals (Broida, 1957; Minkoff, 1959). 

 However, in order to achieve this optimum preservation and effec- 

 tively "fix" the highly reactive free radicals in biological systems, 

 it would be necessary to work at temperatures of liquid helium 

 (Allen, 1952; Mendelssohn, 1956), which are about 100° C lower 

 than those currently obtained with isopentane-liquid nitrogen 

 coolants. 



Improved low-temperature preparation techniques for electron 

 microscopy of biological tissues (Fernandez-Moran, 1959a) have 

 recently been developed which yield better morphological and 

 histochemical preservation of lamellar systems and other cell com- 

 ponents than do the standard freeze-drying or freeze-substitution 

 methods. These "cryofixation techniques" (Fernandez-Moran, 

 1959c) are based on rapid freezing of fresh or glycerinated tissues 

 with liquid hehum II at 1° to 2° K, followed by freeze-substitution 

 and embedding in plastics at low temperatures, under conditions 

 which minimize ice-crystal formation, artificial osmotic gradients, 

 and extraction artifacts. 



The following review deals with salient features of the fine struc- 

 ture of lamellar systems in thin sections of the myelin sheath and 

 selected photoreceptors, as revealed bv high-resolution electron 

 microscopy, using both standard preparation techniques and the 

 new low-temperature procedures. Although the latter are still in a 

 preliminary stage of development, the experimental approaches will 

 be outlined and the underlying operational concepts discussed in 

 order to illustrate the potentialities of ultrastructure research at low 

 temperatures (Fernandez-Moran, 1959d). Lamellar systems are 

 particularly suitable for this type of work, since all steps of the 

 preparation procedures can be followed and the artifact sources 

 analyzed directly at low temperatures bv combined application of 

 x-ray diffraction techniques and electron microscopy. Moreover, 

 by cooling the object during observation in the microscope, and 

 using an electron microbeam of very low intensitv, irradiation 

 damage and specimen contamination can be minimized ( Fernandez- 

 Moran, 1959a, 1959d). With this experimental arrangement for 

 low-temperature electron microscopv, a direct investigation of ice- 



