Morphological study by an 'in vivo cryotechnique' of the shape of erythrocytes circulating in large blood vessels.

Changes in the shape of erythrocytes circulating in large blood vessels of mice were examined by our 'in vivo cryotechnique'. The abdominal aorta and inferior vena cava (IVC) were cut vertically with a precooled knife and simultaneously an isopentane-propane mixture (-193 degrees C) was poured over them for freezing. They were freeze-substituted in acetone containing 2% osmium tetroxide. Some specimens were embedded in Quetol-812, and thick or ultrathin sections were examined by light or transmission electron microscopy. Serial ultrathin sections were used to reconstruct 3-dimensional images of native erythrocytes. Others were transferred into t-butyl alcohol and freeze-dried for scanning electron microscopy. The tissue surfaces were sufficiently frozen to prevent large ice crystal formation, and erythrocyte shapes were also preserved. The shapes of circulating erythrocytes appeared to be varied in the abdominal aorta but typical biconcave discoid shapes were rarely observed. Conversely, erythrocytes were approximately biconcave discoid in shape in the IVC. Our in vivo cryotechnique was useful for clarifying the in vivo morphology of erythrocytes circulating in large blood vessels.