Surface and internal structure correlation: high-voltage and scanning electron microscopies of wholemount alveolar...

We found that the high-voltage electron microscope (HVEM) operating at 1.5MeV was able to transilluminate and form a focused transmission image of whole-mounts of alveolar walls from human lung, a tissue sufficiently thin to require no embedment and sectioning. Resultant micrographs resembled a composite of scanning and transmission electron microscope images: surface and internal structure of the alveolar wall were visualized in a single micrograph. Although the scanning electron microscope extracts some subsurface information in the secondary electron mode, the HVEM produced better images of both surface and subsurface features. Lungs were fixed, dehydrated, critical point dried, and metal coated as for conventional scanning electron microscopy, then individual alveolar walls were excised by hand and mounted on transmission electron microscope grids. Regions of the alveolar wall up to 10 microns thick were delineated with the high-voltage electron microscope. Cell surface characteristics were correlated with cell type as identified by underlying cell internal structure. Whole white blood cells within capillaries of the alveolar wall were identified by the configuration of their nuclei. Features of the nucleus and surface of alveolar type II cells were recorded simultaneously. Whole red blood cells were imaged within intact capillaries that branched and wove from one alveolar surface to the other. HVEM analysis of excised alveolar septa allows definitive correlation of surface and underlying structures in single micrographs of broad portions of the alveolar wall and is an alternative to embedment, microtomy and serial section reconstruction for this uniquely thin tissue.