Scanning electron microscopic study of flowing erythrocytes in hepatic sinusoids as revealed by 'in vivo cryotechnique'.

The purpose of this study is to develop a method for stabilizing erythrocytes under flowing condition in living livers, as revealed by scanning electron microscopy (SEM). After the procedure of the 'in vivo cryotechnique', both freeze-substitution and subsequent t-butyl alcohol freeze-drying methods were used for preparing SEM specimens. By freeze-fracturing with a scalpel in liquid nitrogen before the freeze-substitution, better preserved surface tissues were obtained for examination. Erythrocytes in hepatic sinusoids were clearly detected without plasma components by the freeze-substitution method, and well preserved in parts where they were flowing with their original shapes. Some were accumulated in sinusoids, especially injunctioning areas of sinusoidal networks, as compared with those in narrow lumens between hepatocyte plates. Shapes of such erythrocytes were various, locating along endothelial cells. After stopping the blood supply into livers by artificial cardiac arrest, their shapes were dramatically changed into biconcaves and they became aggregated side by side to be packed in the sinusoids. The three-dimensional shapes of flowing erythrocytes in hepatic sinusoids were demonstrated for the first time by the 'in vivo cryotechnique' combined with SEM.