*4.1. Sample Preparation*

An archival sample of a *human* lung [33], kindly provided by Professor Ewald R. Weibel (Institute of Anatomy, University of Berne, Berne, Switzerland), was used for the current study. The sample was prepared as described previously [6] following a modified protocol from Deerinck et al. [34] to enhance membrane contrast, cf. also [16]:

In brief, the fixed sample was rinsed in 0.15 M HEPES buffer followed by 0.1 M cacodylate buffer and subsequently postfixed by reduced osmium tetroxide (OsO4) (1.5 % hexacyanoferrate II, 1 % OsO4 in 0.1 M cacodylate buffer) in the dark for half an hour. The sample was then washed in double distilled water (ddH2O), infiltrated with 1 % thiocarbohydrazide in ddH2O for 20 min and washed again in ddH2O before it was once more postfixed by 1 % OsO4 in ddH2O in the dark for half an hour. After washing in ddH2O the sample was block stained overnight in the dark at 4 ◦C in an aqueous half saturated uranyl acetate solution, followed by a washing step in ddH2O and block staining by Walton's lead aspartate at 60 ◦C for half an hour. After washing in ddH2O, the sample was dehydrated in an ascending series of acetone (70 %, 90 % and 100 %) and finally embedded in Durcupan (Sigma-Aldrich Chemie GmbH, Munich, Germany).

The sample was trimmed and an ultrathin section (60 nm) was generated to look for appropriate regions of interest in a conventional transmission electron microscope (Morgagni 268, FEI, Eindhoven, Netherlands) for imaging with the FIB SEM.

The Durcupan block was mounted in a slotted SEM specimen holder and the sides were covered with conductive silver (Plano, Wetzlar, Germany) before the sample was sputtered with a 20 nm gold layer (Quorum Q150R ES sputter coater; Quorum Technologies Ltd, Laughton, East Sussex, United Kingdom), cf. [17].

### *4.2. FIB SEM Data Set Acquisition*

The prepared specimen with the region of interest (ROI) was approached with a Zeiss Crossbeam 540 (Carl Zeiss Microscopy GmbH, Jena, Germany) at 20 kV acceleration voltage. A deposition of platinum and the generation of carbon-highlighted marks on the area of interest enabled adequate handling, tracking, autofocus and autostigmation during the acquisition process. Finally, using the Inlens Secondary Electron (SE) and the Energy selective Backscattered (EsB) detector (grid voltage 800 V), a z-stack of 2297 images, each showing a ROI of 33 μm × 20 μm was generated (pixel size 2 nm, section thickness 10 nm). Image acquisition was performed at 1.5 kV with 1.0 nA using the ATLAS software package (Carl Zeiss Microscopy GmbH, Jena, Germany) accompanying the microscope, cf. [17]. For later segmentations and 3D reconstructions a cropped data set was exported from the ATLAS software package with a pixel size of 5 nm and an Inlens SE to EsB detector ratio of 80 % to 20 %.
