Pp2000 t

Small leaf samples of about 5 mm × 5 mm were excised from freshly harvested leaves and immediately fixed on a plane cryo transfer shuttle with conductive mounting medium (1:1 mix of Tissue-Tek and colloidal graphite, Agar Scientific Ltd., Stansted, United Kingdom) and then processed according to [65 (link)]. In brief, after shock-freezing at -210 °C in nitrogen slush, the samples were transferred to a pre-cooled (-135 °C) cryo chamber (PP2000 T, Quorum Technologies Ltd., Laughton, United Kingdom) and sublimated for 15 min at -90 °C for 15 min. After sputtering with platinum (30 s coating at 5–10 mA in an Argon atmosphere), the specimens were transferred to the cryo-stage in the SEM chamber (T = -135 °C) and imaged (Quanta 250 FEG field emission scanning electron microscope, FEI, Brno, Czech Republic) under ultravacuum (3 10^–7 mbar). Backscattered electrons were collected by an Everhart–Thornley detector at a working distance of 5 mm, and an accelerating voltage of 10 kV.

Since the available amount of wax ester oleogel was very low, about 50 mg of the samples were placed on a silicon wafer and partially de-oiled with isobutanol which was poured over the sample. Additionally, individual crystalline platelets from the oleogels were isolated using deposition of a few drops of the retained de-oiling solution (isobutanol with traces of washed out MCT oil) on another silicon wafer. Following freezing by liquid nitrogen, the samples were transferred to the cryo chamber (PP2000 T, Quorum Technologies Ltd., Laughton, UK) and pre-cooled at −135 °C. The samples were sublimated (T = –90 °C, t = 15 min) inside the cryo chamber to eliminate eventual ice contamination on the sample’s surface. The samples were coated with platinum in Argon atmosphere (t = 30 s for coating, current: ca. 5–10 mA). This, in order to minimize charging problems. Lastly, samples were loaded to the cryo stage in the SEM chamber (T = −135 °C). For imaging, Quanta 250 FEG field emission scanning electron microscope (FEI, Brno, Czech Republic) under high vacuum (3 × 10⁻⁷ mbar) was used with an Everhart–Thornley detector (working distance = 5 mm; accelerating voltage = 10 kV). This procedure was kindly performed by the Department of Food Technology and Bioprocess Engineering, Max Rubner-Institut, Karlsruhe, 76131 Germany.