*4.3. Segmentations and 3D Reconstructions*

For segmentations and 3D reconstructions 3dmod (part of the iMOD package [35]) was used: After import of the data set, the cells or cell parts of interest were segmented by manually tracing their outlines on the FIB SEM images. As a basic algorithm the structures were segmented on every fourth image. If the complexity of structure and/or the modeling, however, required a narrower segmentation interval, also sections in between the first and fourth image (up to every image) were segmented. The stack of contours was then used by 3dmod to generate 3D models of the segmented structures. For the AE2 cells also the luminal-abluminal border as an approximation of the apical/basolateral surface border was segmented by tracing the luminal edges of the alveolar cell surface through the z-Stack, cf. [6].

#### *4.4. Figure Preparation*

Figures were prepared in the GNU Image Manipulation Program (gimp 2.10.12; www.gimp.org [accessed on 23 September 2019]) after having created snapshots with 3dmod.

For a general overview of sample preparation techniques and the volume EM workflow, see [10].

**Author Contributions:** (In alphabetical order) Conceptualization, C.M., C.W. and J.P.S.; Methodology, C.W. and J.P.S.; Investigation, C.W. and J.P.S.; Resources, C.M., C.W. and J.P.S.; data curation, C.W. and J.P.S.; writing—original draft, J.P.S.; writing—review and editing, C.M., C.W. and J.P.S.; Visualization, C.W. and J.P.S.; supervision, C.M.; funding acquisition, C.M. and J.P.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work has been supported by the Federal Ministry for Education and Research (BMBF) via the German Center for Lung Research (DZL) and the "Junge Akademie 2.0" of Hannover Medical School, Hannover, Germany.

**Acknowledgments:** The authors wish to thank Susanne Faßbender for excellent technical assistance, David Mastronarde for outstanding support in using IMOD and his efforts to improve the software continuously and Jan Hegermann, Matthias Ochs, Lars Knudsen, Stephanie Groos and Andreas Schmiedl for valuable discussions with impact on the current work. We acknowledge support by the German Research Foundation (DFG) and the Open Access Publication Fund of Hannover Medical School (MHH).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, nor in the decision to publish the results.
