Vct500

As sample material, natural honey from the company Langnese was used. Tungsten wires were used as substrate material and their rough surfaces were produced by applying a tensile force under cryogenic conditions^{43 (link),44 (link),66 (link)}. It’s well known that honey has a high viscosity and, therefore, sticks to different surfaces. In the case of honey, the rough tungsten wire, was initially dipped into the honey and afterwards quenched to cryogenic temperature into a liquid N₂ bath. The obtained honey droplet with a diameter of 50 µm can be seen in Fig. 9B. This method cannot be used for low viscous liquids; therefore, a dedicated method was developed as reported in^{43 (link),44 (link),66 (link)}. After that, the sample holder was transferred as fast as possible into the body of the cooled transfer shuttle VCT500 from Leica (T = − 184 °C). This system allowed to transfer of samples at cryo-temperatures (T = − 184 °C) and under a low-pressure atmosphere (10^–4 mbar) between the machines without contaminations from the environment.Figure 9

FIB Preparation—above in (A) the annular milling process with a decreasing inner diameter and respective current is schematically shown. (B) Frozen honey droplet on top of the tungsten post. (A–I) show the milling process with an annular pattern and decreasing inner diameter until a final tip with a radius < 100–200 nm (J) is obtained.

Trophozoites and cyst-like forms from R. vilicus were observed by microscopy (Olympus IX73 inverted microscope, 1,000x magnification) using a differential interference contrast. All sizes were measured using ImageJ (Schneider et al., 2012 (link)). The morphological comparison between trophozoite and cyst-like forms was also assessed by electron microscopy. For scanning electron microscopy (SEM), cells were cryofixed in nitrogen with VCM (Leica), sublimed and 4 nm wolfram coated in ACE 600 (Leica). Each step was performed under vacuum and samples were transferred from one station to another using VCT 500 (Leica). Samples were observed at −100°C in scanning electron microscope Teneo VolumeScope (Thermo Fisher) to 3.5 kV and 10 kV.
For transmission electron microscopy (TEM), amoebae were fixed with 2.5% glutaraldehyde and 2% osmium tetroxide. Dehydration was carried out using different concentrations of acetone. Epon resin were used for impregnation and embedding process. After cutting with ultramicrotome UC6 (Leica), samples were contrasted with uranyl acetate and lead citrate. Samples observations were proceeded on transmission electron microscope (JEOL 1010) at 80 keV. Images were acquired with Quemesa camera (Olympus).

Using a custom-made atom probe^{56 (link)} equipped with NOPA to continuously change wavelength in the range between 350 and 900 nm, measurements were conducted with a laser wavelength of 355 nm. The pulse length was chosen to 250 fs. The spot size in the focus point amounts to a diameter of 50 microns. As detector, a 120 mm diameter delay line detector with chevron MCP setup with an open area ratio (OAR) of 50% is used. The flight length amounts to 130 mm and the effective half angle of the field of view to 38°. The system is equipped with a custom-made cryo-transfer port to accept a standard VCT500 from Leica for the transfer of cryogenic samples. The exchange of the sample in the buffer chamber uses a PEEK isolated storage position to avoid melting of the sample during transfer. The obtained datasets were analysed and reconstructed using the Scito⁵⁷ software package. Calculated mass spectra were exported into csv files and plotted using OriginPro⁵⁸ for publication.

High-pressure freezing, fracture, and cryogenic SEM imaging were performed to examine the inner structure of cellulosic biofilms formed at the various interfaces. The cellulosic biofilm samples were placed in specimen carriers and mounted on a sample holder. The samples were then frozen under high pressure using a high-pressure freezing system (Leica EM HPM100, Leica Microsystems Inc., Austria). This was followed by fracturing at -110 °C using a flat edge knife and sublimation at -95 °C for 5 min under the vacuum of 10^-7 mbar in a freeze-fracture and etching machine (Leica EM ACE900). The samples were transferred to the SEM via a cryo-transfer system (Leica EM VCT500). High-vacuum secondary electron imaging was performed using an Apreo VS SEM at 500 V. During SEM imaging, the chamber temperature was maintained at -110 °C.

The presented results were obtained by using a custom-made atom probe^{68 (link)} operating at a laser wavelength of 355 nm. The pulse length accounts for 250 fs and the spot size diameter to 50 microns. The system is equipped with a 120 mm diameter delay line detector with an open area ratio (OAR) of 50%. The system was additionally equipped with a custom-made cryo-transfer port to accept a standard VCT500 from Leica for the transfer of cryogenic samples.