The Three-Dimensional Ultrastructure of the Human Alveolar Epithelium Revealed by Focused Ion Beam Electron Microscopy

Abstract

Thin type 1 alveolar epithelial (AE1) and surfactant producing type 2 alveolar epithelial (AE2) cells line the alveoli in the lung and are essential for normal lung function. Function is intimately interrelated to structure, so that detailed knowledge of the epithelial ultrastructure can significantly enhance our understanding of its function. The basolateral surface of the cells or the epithelial contact sites are of special interest, because they play an important role in intercellular communication or stabilizing the epithelium. The latter is in particular important for the lung with its variable volume. The aim of the present study was to investigate the three-dimensional (3D) ultrastructure of the human alveolar epithelium focusing on contact sites and the basolateral cell membrane of AE2 cells using focused ion beam electron microscopy and subsequent 3D reconstructions. The study provides detailed surface reconstructions of two AE1 cell domains and two AE2 cells, showing AE1/AE1, AE1/AE2 and AE2/AE2 contact sites, basolateral microvilli pits at AE2 cells and small AE1 processes beneath AE2 cells. Furthermore, we show reconstructions of a surfactant secretion pore, enlargements of the apical AE1 cell surface and long folds bordering grooves on the basal AE1 cell surface. The functional implications of our findings are discussed. These findings may lay the structural basis for further molecular investigations.

Volume electron microscopy: analyzing the lung

“…Conventional SEMs can reveal cellular surface details but cannot look into the cell simultaneously. (Single section) TEM reveals intracellular details, but may fail to transport important topographic information like the belonging of certain profiles (see, Rogers and Haller 1978 ; Schneider et al 2019 , 2020 ). The 3D information gained by volume EM, however, allows scientists to see arbitrary section planes in EM resolution and to generate 3D models that can be viewed from any angle with variable transparency.…”

“…A recent study on the human alveolar epithelium demonstrates the benefit of FIB-SEM for the visualization of fine structural details: FIB-SEM could image an almost complete AE2 cell and enable detailed reconstructions of a LB secretion event, the microvilli lawn on the cell surface and tiny AE1 cell processes beneath AE2 cells (Schneider et al 2020 ). Also subcellular details in AE2 cells (plate bodies) could be revealed in 3D using FIB-SEM (Mühlfeld et al 2020 ).…”

“…Traditionally, conventional microscopy, however, is faced with the problem that thin sections of the tissue of interest have to be prepared, which disassembles its three-dimensional (3D) nature (for transmission electron microscopy (TEM), in general, 60–90 nm thin sections are used (Hoppert 2003 )). Indeed, (single section) TEM can reveal fine structural details, such as Weibel–Palade bodies in the endothelium (Weibel 2012 , 2017 ) or single lipid lamellae in lamellar bodies (LBs) of AE2 cells (Ochs 2010 ), but it may fail to transport important spatial information, like the belonging of two separate cell profiles to the same cell [examples are found in Schneider et al ( 2019 , 2020 )].…”

The plate body: 3D ultrastructure of a facultative organelle of alveolar epithelial type II cells involved in SP-A trafficking

“…To increase the volume, the tomographic visualization shown in this manuscript is based on tomograms of four consecutive 300 nm thick sections. A relatively new method is FIB-SEM which has only rarely been used in lungs so far (Købler et al 2014 ; Ochs et al 2016 ; Hegermann et al 2019 ; Schneider et al 2020 ). The x – y resolution does not reach the levels of TEM tomography but allows visualization of larger volumes (whole cells) with a z resolution of approximately 10 nm.…”

The History and Mystery of Alveolar Epithelial Type II Cells: Focus on Their Physiologic and Pathologic Role in Lung

“…Several authors provided insights as to the topological complexity of the alveolar epithelium by serial block face scanning electron microscopy (SBF-SEM), also showing how an extremely broad and thin gas exchange surface (130 ± 12 m 2 alveolar surface, 115 ± 12 m 2 capillary surface, 0.62 ± 0.04 ηm 2 double monolayer tissue barrier thickness) is forced into a limited space, thanks to a fractal folding forming crumpled space-filling continuum of alveoli lined to very thin capillaries and open to airways [ 22 , 23 , 24 ].…”