*2.3. Epithelial Surface*

The apical AE2 cell surface exhibits smooth areas surrounded by microvilli as described previously [1]. The pink cell has two larger plain areas and one of them shows an open surfactant secretion pore (Figure 2). (At this stage of exocytosis) the pore has a smaller diameter than the vesicle under secretion, cf. [1]. The green AE2 cell shows a larger plain surface area in the recess between the adjacent AE2 cells (Figure 1). Also the basolateral AE2 cell surface shows abundant microvilli. In contrast to the luminal microvilli, they may appear clustered in small groups, located in small niches of the cell surface. These microvilli are found both between adjacent epithelial cells (AE2 and AE2 or AE2 and AE1), but also at gaps in the basal lamina, where they may reach interstitial cells (Figure 3). They are even found above a continuous basal lamina (not shown).

The AE1 cell domains also exhibit a combination of smooth surface areas with surface enlargements (Figures 1 and 4). In contrast to the AE2 cells, this enlargement is realized primarily by protrusions of the plasma membrane filled with cytoplasm. However, some microvilli are found here as well, which can be identified by their regular structure and inner architecture of the cytoskeleton. Some microvilli may share a common basis and some seem to rest on surface protrusions. Both AE1 cell domains show grooves bordered by cellular folds on their basal surface. These grooves stretch across the cells in a relatively orientated fashion along space. Beneath the groove of the yellow AE1 cell domain primarily extracellular matrix is found while it is primarily capillary endothelium beneath the blue cell domain (Figure 5). Additionally, the AE1 cells show abundant caveolae and it is the basal compartment that seems to house most of them (Figure 5B).

**Figure 2. Luminal AE2 cell surface and surfactant secretion pore.** (**A**) Detail from image 884 which shows an open surfactant secretion pore (arrowhead) and remnants of a lamellar body inside (asterisk). In this image the opening is much smaller than the profile diameter of the vesicle under secretion. (**B**) 3D model of the AE2 cell with the secretion pore (arrowhead) and the remnants of the lamellar body inside (yellow, asterisk). Note the plain cell surface in this area. (**C**) Same image content as in B but with a transparent cell body, which reveals the entire remnant of the lamellar body and indicates the size of the secretory vesicle (circumference marked by arrowheads), the projection of which is much larger than the secretion pore. Scale bars: 1 μm. The black lines in B and C indicate the position of the section plane of A.

**Figure 3. Basolateral surface of AE2 cells.** (**A**) Segmented electron micrograph (image 988 of the data set). The micrograph shows the segmentations of the pink and green AE2 cell as well as of the yellow AE1 cell domain with opaque outlines and transparent filling. Microvilli can be found at different sites of the AE2 cell, three of them are indicated on the micrograph: towards the adjacent green AE2 cell, which also shows microvilli at this site (filled arrowheads 2 and 3), towards the yellow AE1 cell domain (filled arrowhead 4), towards an interstitial cell (hash key) through a hole in the basal lamina (filled arrowhead 5). Turquoise circles indicate the lateral luminal cell border of the pink AE2 cell (empty arrowheads). The position at empty arrowhead 1 is found in the 3D model in C. The dotted line indicates the profile of the groove depicted in B. (**B**) 3D Model of the pink and green AE2 cells. The position of the segmented electron microscopic (EM) plane in A is indicated in black and the lateral luminal cell borders of the pink and green AE2 cells are indicated by turquoise and blue delineations, respectively (empty arrowheads; note that the positions do not correspond to the empty arrowheads in A). The pink AE2 cell has only one luminal surface. Note the abrupt appearance of a dense microvilli lawn on the luminal surface in some areas (asterisk). Filled arrowhead 5 indicates the region of the corresponding arrowhead 5 in A. Note also the surrounding basolateral microvilli, which may appear in larger groups. Two sites of basolateral groups of microvilli on the green cell are indicated (unnumbered filled arrowheads). Note the long and mostly smooth groove of the pink AE2 cell surface (dotted line). (**C**) Opaque 3D model of the pink AE2 cell and transparent models of the green AE2 cell and the yellow AE1 cell domain. The position of the segmented EM plane in A is indicated in black. The transparency of the yellow AE1 cell domain and the green AE2 cell enable visualization of intercellular microvilli. Numbered arrowheads correspond to the arrowheads in A: Filled arrowhead 2 and 3: Microvilli between the pink and green AE2 cells. Filled arrowhead 4: AE2 microvilli towards the yellow AE1 cell domain. The unnumbered filled arrowhead indicates another region with AE2 microvilli under the yellow AE1 cell domain. The lateral luminal AE2 cell borders are indicated by turquoise and blue delineations (cf. B). Empty arrowhead 1 refers to the corresponding empty arrowhead in A. Black arrows indicate the small parts of the cell where the cell extends beyond the available dataset. Scale bars: 1 μm.

**Figure 4. Luminal surface of AE1 cells.** (**A–C**) Images 1172, 1348 and 1520 of the dataset. Filled arrowheads 1 and 2 (**A**) indicate profiles of microvilli. Filled arrowheads 3 and 4 (**B**) indicate microvilli with a shared basis. Empty arrowheads 5 and 6 (**A**) and 7–10 (**C**) indicate profiles of plasma membrane protrusions filled with cytoplasm. The hash key indicates a region with smooth AE1 luminal surface. (**D**) Detail of the 3D model. Arrowheads and hash key refer to the particular profiles in A–C. The particular section planes 1172, 1348 and 1520 are labeled and emphasized by black color in the model. The different thickness of these black markings is caused by different segmentation intervals in these regions. Note the smooth surface of the cell in the center of the image and the surface enlargements around. Some of them seem to be concentrated in a surface cavity (asterisk). Scale bars: μm.

**Figure 5. Basolateral surface of AE1 cells.** (**A**) Detail of the segmented EM plane 1384 including parts of the yellow AE1 cell domain. At the basal side, small folds may appear which create a groove-like profile with the concavity pointing towards the interstitial space. The groove beyond the epithelium is filled with extracellular matrix (arrowhead 1). (**B**) A similar situation is found at the basal side of the blue AE1 cell domain, but here the groove is filled with capillary endothelium (arrowhead 2). Note the densely packed caveolae, which appear to be predominantly located in the basal compartment of the cell (asterisk). (**C**) View on the basal side of the 3D model. The model reveals that indeed the AE1 cell domains form grooves on their basal side, which are bordered by cellular folds. The groove in the AE2 cell surface, which has been already described in Figure 3, is also indicated by a dotted line. The EM plane 1384 is indicated by black color. Scale bars: 1 μm.

#### *2.4. Sites of Cell Contacts*

The AE1 cell domains contact each other with or without overlap. At a certain point, they may simply prod against each other (edge to edge), while a few micrometers further one cell domain slips under the other. Directly at the site of contact they may bulge out into the alveolus (Figures 6–8). On the basolateral side, cellular processes may interdigitate with the neighbor cell domain, while the luminal edges appear more regular (Figure 9).

At the contact site to AE2 cells, AE1 cells may simultaneously both crawl up and beneath AE2 cells. This is achieved by a y-shaped branching pattern with one branch crawling up and the other branch crawling beneath the AE2 cell. In 3D, this behavior reminds of a bowl partially enwrapping the AE2 cell (Figure 10). Additionally, the AE1 cells may send out thin, finger-like processes into the space between the AE2 cells and the underlying basal lamina. Such processes can be found in close proximity to AE2 microvilli (Figure 11).

The two AE2 cells contact each other edge to edge, with niches of microvilli facing each other (Figure 12).

**Figure 6. AE1/AE1 contact.** (**A**–**D**) show the x-plane reconstructions 1603, 1987, 2179 and 2251 of the data set (lateral view on the dataset) on the left side of the panel and a clipped model of the yellow and blue AE1 cell domains on the right side of the panel. The transect plane in the foreground corresponds to the EM plane on the left side. The site of cell contact is indicated by arrowheads. The distance between the different transect planes is indicated in micrometers between the models. Note how the yellow AE1 cell domain slips under the blue cell domain in A and C while the cell domains in B and D meet each other just edge to edge. Note also the bulging of the cell domains into the alveolar lumen at the contact site (asterisks on the EM images). Scale bars: 1 μm.

**Figure 7. AE1/AE1 contact.** (**A**,**B**) show the yellow and blue AE1 cell domain with their alveolar surfaces. On each image one of both cell domains is shown transparently (A: yellow, B: blue) to visualize overlap. Images (**C**,**D**) follow the same principle but show the basolateral surfaces. Scale bars: 1 μm.

**Figure 8. AE1/AE1 contact.** The image shows the contact of parts of the yellow and blue AE1 cell domains. The blue cell domain in the foreground is displayed transparently to enable visualization of the yellow AE1 cell domain. Note how the yellow AE1 cell domain slips beneath the blue cell domain. Scale bar: 1 μm.

**Figure 9. AE1/AE1 contact.** (**A**,**B**) again show the yellow and blue AE1 cell domain from a luminal (**A**) and a basolateral (**B**) perspective but without transparency. Note the irregular contact site on the abluminal side compared to the luminal side caused by interdigitating cell processes. Compare in particular the region emphasized by the ellipse. Scale bars: 1 μm.

**Figure 10. AE1/AE2 contact.** (**A**) 3D model of the two AE1 cell domains and AE2 cells. AE2 cell models are transparent to enable visualization of the yellow AE1 cell domain behind the AE2 cells. AE1 cells may branch at the edge of an AE2 cell and extend their branches beneath and over the AE2 cell to form a bowl-like structure that enwraps the AE2 cell partially. The yellow AE1 cell domain forms a bowl at the edge of the pink AE2 cell (behind the transparent AE2 cell model). Note also the y-shaped branching of the blue AE1 cell domain and its extension beneath the pink AE2 cell (arrow). The black line indicates the position of the transect image in B. (**B**) Reconstruction of the y-plane (lateral view on the dataset) at the position indicated by the black line in A. The profiles of the yellow AE1 cell domain and the pink AE2 cell are indicated by yellow and pink outlines. Note the y-shaped profile of the yellow AE1 cell domain. The surface of the yellow AE1 cell domain ("inner surface" of the bowl) looked at in A and C is indicated by arrowheads. (**C**) 3D model of the yellow and blue AE1 cell domain transected by the y-plane shown in B. The different angle of view compared to A facilitates the imagination of a bowl formed by the yellow AE1 cell domain. The arrowhead indicates where the image plane transects the bowl of the yellow AE1 cell domain. Scale bars: 1 μm.

**Figure 11. AE1/AE2 contact.** (**A**) Detail of segmented image 1208. The image shows the segmentation of the pink and green AE2 cells as well as of the yellow and blue AE1 cell domain. Note the very small profiles of the yellow AE1 cell domain (arrowheads 1–5) and the small profile of the blue AE1 cell domain (arrowhead 6). These will probably be overseen if only single images are investigated. If they are recognized, it will almost be impossible to assign them to different cells (compare B). Only the sequence of images reveals their belonging. (**B**) The same EM image as in A but without segmentations. (**C**) 3D Modell of the yellow and blue AE1 cell domain as well as the pink and green AE2 cell with a view on the basolateral cell surfaces. The 3D model reveals that the labeled profiles in A belong to small and thin AE1 processes that crawl along the surface of the pink AE2 cell. The numbered arrowheads correspond to the arrowheads in A. Note also the processes of the yellow cell domain along the green AE2 cell. Some of the processes are found in close proximity to AE2 cell microvilli (asterisks). Scale bars: 1 μm.

**Figure 12. AE2/AE2 contact.** (**A**) EM image 1112. The image shows the green (top) and pink (bottom) AE2 cell contacting each other. Both cells show niches with microvilli on their basolateral surface facing each other (filled arrowheads). Between both cells also parts of the yellow AE1 cell domain are found (asterisk). The alveolar lumen is indicated by the hash key and the basal lamina by an empty arrowhead. (**B**) 3D model of the pink and green AE2 cell as well as the yellow and blue AE1 cell domains. The image plane of A is indicated in black. Arrowheads correspond to the arrowheads in A. The borders of the luminal/abluminal surface of the AE2 cells are indicated by turquoise (pink cell) and blue lines (green cell) (cf. Figure 3). The pink AE2 cell and its luminal/abluminal border are displayed transparently to visualize the green AE2 cell and yellow AE1 cell domain behind as well as the luminal/abluminal border of the green AE2 cell. Note the well defined niches with microvilli on the basolateral surface of the green AE2 cell (black arrowheads). Scale bars: 1 μm.

#### **3. Discussion**

Volume electron microscopic (EM) techniques for 3D modeling of biological structures, including "conventional" techniques like single sectioning transmission electron microscopy (ssTEM) and "new" techniques like SBF SEM and FIB SEM, have been reviewed extensively recently, e.g., [9–13] and some of them have already been used for 3D reconstructions of lung structure: ssTEM [5,7,8,14]; electron tomography [15]; array tomography [16] and SBF SEM [6,11]. Even FIB SEM has been applied [17–19], but the study of Købler et al. was rather focused on testing an alternative method to diamond knife ultramicrotomy and transmission electron microscopic imaging of nanotubes; the review of Ochs et al. gave an outlook on the potential of this technique in lung research; and Hegermann et al. demonstrated a method for correlative light and electron microscopy. Kremer et al. [11] showed a FIB SEM-based reconstruction of a small part of a *human* A549 cell. Here, to our knowledge, we provide the first extensive study of the alveolar epithelial 3D ultrastructure based on FIB SEM in a *human* lung sample. We were able to reconstruct an almost complete AE2 cell with two adjacent AE1 cell domains as well as a part of a neighboring AE2 cell. The model shows detailed reconstructions of the epithelial surface, including a surfactant secretion pore on an AE2 cell, enlargements of the apical AE1 cell surface, long folds bordering grooves on the basal AE1 cell surface, AE1/AE1, AE1/AE2 and AE2/AE2 contact sites, basolateral microvilli pits at AE2 cells and small AE1 processes beneath AE2 cells. The functional relevance of these findings will be discussed below.

AE1 cells seem to be responsible for the majority of fluid transport across the alveolar epithelium [20–22]. According to Dobbs et al. [20] different sodium channels (highly selective Na<sup>+</sup> channel (HSC), non-selective Na<sup>+</sup> channel (NSC) and cyclic nucleotide-gated channel (CNG)), the cystic fibrosis transmembrane regulator (chloride channel), potassium and water (Aquaporin 5) channels and Na+/K+ATPases are involved in that. The surface enlargements of the AE1 cell domains shown here may serve as a membrane compartment housing the particular transport proteins and, thus, be the ultrastructural correlate of liquid absorbtion.

Interestingly, when looking at Figure 4, one gets the idea, that the surface in a septal concavity is rich in these surface enlargements, while they seem to be reduced or even absent on convexities. Concave niches are filled with the (liquid) hypophase of surfactant [1,7] and these "puddles" may be the sites where most of the fluid absorption takes place. Microvilli on AE1 cells have been described previously [20] and were also found during *cat* lung development [8,14].

Fixation (cf. the studies of Gil et al. [23], Oldmixon et al. [24] and Oldmixon and Hoppin Jr. [25] or long-term storage can lead to microscopic artefacts. The following arguments, however, indicate that the non-microvillus surface enlargements are a real biological phenomenon: (1) The cytoplasm within these excrescences is quite electron dense, while lytic cell parts regularly appear electron light. (2) The surface enlargements seem to be concentrated at certain spots, while other areas remain smooth. (3) Microvillus-like surface irregularities without obvious microfilaments have also been described by Mercurio and Rhodin [7] in the *cat*. (4) Surface irregularities on AE1 cells can also be seen on some scanning electron micrographs in textbooks or other articles [1,3,5,26,27].

Another explanation for the surface irregularities was given by Mercurio and Rhodin [7], who suggested that they are incorporated into the cell membrane during inflation. This was concluded, because they also found a cell with a rather smooth surface, where they thought that the flatness was caused by trapped air in the airspaces. If this interpretation is true, however, then also the basolateral cell membrane needs membrane reserves, since the apical and basolateral membranes are separated by tight junctions [28]; Sirianni et al. [5] also mentioned the existence of discontinuous tight junctions. Such reserves could be provided by the cytoplasmic folds of the basolateral cell membrane or by the AE1 cell branches beneath the AE2 cells shown by our models.

The basal AE1 cell membrane folds, however, could also serve as mechanical stabilization, i. e., anchoring the epithelium in the septal wall by interdigitation with the interstitium to resist shearing forces. This principle is well established for the skin, where connective tissue papillae or ridges of the dermal papillary layer interdigitate with the epidermis at the dermal-epidermal junction [29] (p. 147).

With respect to AE1/AE1 cell contacts in the *cat* lung, Mercurio and Rhodin [7,8] described the possibility that adjacent cells may show alternating overlap or meet edge to edge. Our data suggest that this is also true for the *human* lung, even if the images and models shown here only demonstrate a clear overlap with the blue cell domain on top of the yellow cell or edge to edge contacts (Figures 6 and 7). What is the functional relevance of this overlap? Overlap per se enhances mechanical stability but additionally enlarges the contact area; in particular it may enlarge the available "contact area" for important cell junctions like the relatively wide (see [1,30]) tight junctions and/or gap junctions, for review, see [31]. The latter have been described to be in close topographic relation to the occluding junctions [30]. Mercurio and Rhodin [7] suggested that the overlap may change during breathing.

With respect to the AE1/AE2 contacts, we could confirm and extend our SBF-based presumption and Sirianni et al's [5] data that AE1 cells may extend both under and on the AE2 cells. Our data set and model revealed a y-shaped branching pattern in 3D (cf. Figure 2 of [5]) and additionally added that AE1 cells may extend various thin foot processes under AE2 cells.

Branches beneath AE2 cells may serve as membrane reserve during inspiration (see above). This may also be an explanation for the foot processes, but these may also be structural correlate of cellular interaction: Sirianni et al. [5] demonstrated linkage of AE1 and AE2 cells with interstitial fibroblasts via holes in the basal lamina, which seem to be primarily located beneath AE2 cells and Nabhan et al. [32] demonstrated that Wnt signaling between fibroblasts and alveolar epithelial stem cells (a small fraction of distinct AE2 cells) is necessary to maintain their stem cell status. Eventually, the small AE1 processes "look for" those holes for linkage to interstitial fibroblasts. Alternatively, the close proximity of these processes to AE2 microvilli pits in some cases (see Figure 11) could indicate exchange of material (too large for gap junctional transport) from AE1 to AE2 cells or vice versa (or fibroblasts).

These microvilli pits of AE2 cells, however, were also found towards other cell types (i.e. the other AE2 cell or interstitial cells (see Figure 3). Interestingly, they were also found above an intact basal lamina (not shown). Proximity to cells and holes in the basal lamina suggests a role in intercellular communication, material exchange or sensory functions. Localization above a closed basal lamina may be the structural correlate of basal lamina turnover or the initiation of creating a hole for consecutive subepithelial communication.

Summing up, the current FIB SEM study of a *human* lung sample provided new insights into the *human* alveolar epithelial cell morphology and topography. Our model reveals detailed reconstructions of the alveolar epithelial surface, including a surfactant secretion pore on an AE2 cell, enlargements of the apical AE1 cell surface, long folds bordering grooves on the basal AE1 cell surface, AE1/AE1, AE1/AE2 and AE2/AE2 contact sites, basolateral microvilli pits at AE2 cells and small AE1 processes beneath AE2 cells. These data may serve as morphological blueprint for molecular investigations of alveolar epithelial biology.

### **4. Materials and Methods**
