FIG. 5.

Freeze substitution of fractured cryovials after cooling at a linear rate of 1°C/min. Ultrastructure resulting from controlled ice nucleation (A, C) is compared with spontaneous ice nucleation (B, D). In spontaneously nucleated samples, the spaces originally occupied by ice crystals are revealed as voids following sublimation of ice, and can be seen throughout the ELS structure. These ice voids are absent within cholesterol-nucleated ELS, although there is evidence of shrinkage spaces. At higher magnification, it is difficult to distinguish cell organelles in cholesterol-nucleated ELS due to the extreme cell dehydration during cooling. Conversely, organelles can be seen in spontaneously nucleated ELS. Cellular material (c), ice voids (i), cell membrane (cm), extracellular matrix (ecm), mitochondria (m) shrinkage spaces (s), and alginate (*) have been labeled. Scale bars on (A, B) 2 μm and on (C, D) 500 nm.