**1. Introduction**

Type 1 alveolar epithelial (AE1) and type 2 alveolar epithelial (AE2) cells form the epithelial lining of alveoli in the *human* lung. Both are essential for normal lung function: AE1 cells cover the majority of the alveolar surface with thin cytoplasmic extensions that participate in a very thin blood gas barrier and AE2 cells serve as the regeneration source for the alveolar epithelium and secrete the pulmonary surfactant, which lowers the surface tension at the air liquid interface and, thus, prevents alveolar collapse [1], see also [2]. In the past, it was suggested that these cells may show complex morphological properties, such as having more than one apical surface for serving different alveoli or branching of AE1 cells [3,4]. Sirianni et al. [5] described basolateral microvilli on AE2 cells for contacting interstitial fibroblasts and AE1 cells that extend both above and underneath AE2 cells. In a recent study, three entire AE1 cells were modeled in three dimensions (3D) by manual segmentation of a serial block-face

(SBF) scanning electron microscopic data set of a *human* lung sample. Both the branching capability and the chance of having more than one apical surface could be confirmed for AE1 cells by this method. Additionally, the 3D data set revealed that different branches of the same cell can form cellular junctions between each other [6]. During this study the questions arose how the sites of contacts both between AE1 and AE1 and between AE1 and AE2 cells as well as the lateral borders of AE2 cells are configured in 3D. The SBF data suggested a complex morphology of AE1/AE2 cell contacts, variable overlap of adjacent AE1 cells and the existence of numerous microvilli that may appear clustered in small niches of the basolateral AE2 cell surface, findings in line with those of Sirianni et al. [5] and Mercurio and Rhodin [7,8]. A special 3D organization of the contact sites may be of relevance for the mechanical stability (or behavior) and integrity of the alveolar epithelium during breathing or the migration of cells from the interstitial space into the alveolus. Knowledge about the detailed 3D organization of the basolateral cell membrane of AE2 cells may help to understand the crosstalk between AE2 and other cells of the septal wall.

While the SBF scanning electron microscope (SEM) used by Schneider et al. [6] has the advantage that it can image rather large fields of view (FOV) and, thus, volumes with entire AE1 cells, it has to live with a compromise in lateral and axial resolution. A high resolution combined with a large FOV tremendously increases the scanning duration (see [9]) and/or may lead to beam damage at the specimen because of exposure to the electron beam. The latter is in particular significant for lung samples because of the small amount of conducting tissue in the epoxy resin block since the lung primarily "consists of air". Using lower section thicknesses fortifies this problem, cf. [10], so the minimal section thickness as the major determinant of the z-resolution is also a limiting factor of SBF SEM. Thus, for 3D evaluation of structural details, such as cellular junctions or microvilli, focused ion beam (FIB) SEM with both a higher x-/y- and in particular higher z-resolution, appeared to be more appropriate (consider the dimensions of microvilli and the section thicknesses in the aforementioned studies: 80 nm in Schneider et al. [6], 100 nm in Sirianni et al. [5] and unknown in Mercurio and Rhodin [7,8], for review of SBF SEM and FIB SEM, see [10–12]). As a consequence, after having reconstructed entire AE1 cells by SBF SEM, we conducted the current study to explore the complexity of the AE1 cell contact sites and the structure of the basolateral AE2 cell membrane using FIB SEM. It was expected that the new insights into AE1 and AE2 cell ultrastructure would enhance our functional understanding of the alveolar epithelium.

### **2. Results**

#### *2.1. Generation of the Data Set for 3D Reconstructions*

After export, the data set comprised 2297 images with a size of 6663 × 4635 px<sup>2</sup> and a pixel size of 5 nm. Based on the number of images and the penetration depth into the z-direction, an average section thickness of 9.94 nm could be calculated, which is less than 1 % deviation from the desired thickness of 10 nm. Since the milling process and, thus, the section thickness, is an undulation around an average and because the system needs a stabilization period after initiation of the image acquisition, the first 139 images were not accounted for in the calculation. This cut off was determined by qualitative inspection of the z-advance at the beginning of the data set.

#### *2.2. Segmentation and 3D Reconstructions*

Two AE1 cell domains and parts of two AE2 cells in immediate topographic relationship were segmented and reconstructed in 3D for the current work. The part of the dataset underlying the segmentations comprises the images 378 to 1624 of the dataset. A global overview of the models is given in Figure 1 and Table 1 indicates how many outlines per AE1 cell domain and AE2 cell were manually segmented (12,623 in total). It should be noted at this point, that AE1 cells are obviously capable of making cellular junctions with themselves [6], so it cannot be excluded that the two "AE1 models" are parts of one and the same cell. To account for this, we stick to the term AE1 cell domain(s) instead of AE1 cell(s), when we refer to the particular models. Apart from a few, very small portions, the pink AE2 cell is completely included in the dataset and, thus, the model also comprises almost the entire AE2 cell. The small "defects" because of missing portions can be seen in Figure 3.

**Figure 1. Overview of the 3D model.** The figure shows an overview of the 3D model from an alveolar viewing direction. The model includes an almost complete type 2 alveolar epithelial (AE2) cell (pink) and parts of another AE2 cell (green) as well as two type 1 alveolar epithelial (AE1) cell domains (blue and yellow), which are labeled as AE2 and AE1, respectively. The filled arrowhead indicates a deep recess between the pink and green AE2 cells as well as the yellow AE1 cell. The AE2 cells show abundant microvilli on their surfaces but also plain parts (empty arrowhead on the pink AE2 cell, for example). The luminal AE1 cell surface may also be plain at a certain spot (asterisk) or enlarged somewhere else (hash key). Scale bar: 1 μm.


