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A morphological approach to cell dynamics is usually difficult, since routine preparative techniques for
electron microscopy always induce artifacts due to cessation of the blood supply into organs. An in vivo
cryotechnique followed by the freeze-substitution method probably reduces such problems. It was applied
for examining the pulmonary alveoli of BALB/c mice in vivo. The following ultrastructural features were
revealed. (1) A surfactant layer provided a continuous covering to the alveolar epithelium. (2) Pleural
epithelial cells, alveolar cells and endothelial cells contained many small vesicles and pits. In the alveolar
epithelium, they were often localised near microtubules. (3) Typical lamellar structures in large alveolar
epithelial cells were rarely detected. (4) Circulating erythrocytes with various shapes were observed in
branching blood capillaries. (5) A close association between erythrocytes and the endothelium was seen at
the peripheral alveolar septum. Such ultrastructural arrangements may be appropriate for the physiological
functions of the pulmonary alveoli, such as exchanges of gases or materials in vivo.
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°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.
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