For preparing the common “biopsy” and immersion-fixed specimens for light microscopy , extracellular and intracellular components were easily leaked out or moved with mechanical compression forces or dehydration and embedding steps [19]. In the CB specimens, PAS -reaction product s were strongly and homogeneously recognized in the cytoplasm of hepatocytes (Fig. 47.1f–h). However, they were heterogeneously observed in the conventional quick-freezing (QF) liver hepatocyte cytoplasm (Fig. 47.1i, small arrows). On the other hand, by conventional immersion fixation and dehydration samples, PAS-reaction products were weakly observed in most of hepatocyte cytoplasm (Fig. 47.1j, small arrows). So, the CB technique would be a useful tool for examining soluble component s .

By in vivo cryotechnique , the sinusoidal erythrocytes could be cryoimmobilized in vivo with various shapes in blood vessel s , as reported before [10, 11]. At the initial contact surface of freeze-fractured CB liver tissues, some erythrocytes were compactly localized in the hepatic sinusoids and the Disse’s space was not clearly open (Fig. 47.2a). To the contrary, open hepatic sinusoids and flowing erythrocyte s were clearly observed in slightly deeper areas with Disse’s spaces (Fig. 47.2b). The morphology of freeze-fractured hepatocytes showed nuclei, many globular mitochondria, and endoplasmic reticulum in their cytoplasm (Fig. 47.2c). At higher magnification, freeze-fractured mitochondria (M) and endoplasmic reticulum (ER) were clearly observed in the hepatocytes (Fig. 47.2c, inset). The Disse’s spaces were covered with sinusoidal endothelium and hepatocyte surface with microvilli (Fig. 47.2c, asterisk). In some areas of Disse’s spaces, hepatocyte cell membranes were slightly separated from each other, which reached deeper near bile canaliculi (Fig. 47.2c, small arrows). The CB samples applied to freeze-fracture technique for SEM, functioning three-dimensional structure, would be examined in living animal livers.