Morphological analysis of lamellar structures in mouse type II pneumocytes by quick-freezing and freeze-drying with osmium tetroxide vapor-fixation

The lamellar body is a membranous structure periodically laminating in vesicles that is known as the most distinctive feature of type II pneumocytes by conventional preparation methods for transmission electron microscopy. The quick-freezing and freeze-drying method, followed by osmium tetroxide vapor-fixation (QF-FD-OsV), was performed to examine the in situ morphology of the lamellar body in type II pneumocytes of living mouse lungs. Typical lamellar structures were rarely seen in vesicles of the type II pneumocytes, but amorphous components and dispersed stripes were often detected in the vesicles, as revealed by the QF-FD-OsV method. To clarify how the lamellar body was formed during the conventional preparation steps, lung tissues of mice were treated with different fixation procedures, such as immersion-fixation with osmium tetroxide or perfusion-fixation with glutaraldehyde followed by osmium tetroxide, in combination with alcohol dehydration or QF-FD-OsV. In addition to lamellar bodies of type II pneumocytes in the specimens with alcohol dehydration, some lamellar structures were also formed even with the QF-FD-OsV method. These findings suggest that the labile lamellar body is easily modified and formed during both chemical fixation and alcohol dehydration steps.