2.3.5 Environmental scanning electron microscope
Surface characterization with light microscopy was complemented by
environmental scanning electron microscopy (ESEM). ESEM differs from
conventional SEM in that only a low vacuum is applied to the chamber,
which allows for rather high humidity, while a high vacuum can be
maintained in the column (McGregor and Donald 2010; Stabentheiner et al.
2010). Therefore, fully hydrated specimens can be imaged under
near-native conditions without the need for sample preparation, which
often results in technical artefacts and morphological alterations (Yuan
et al. 2020). Fresh leaf samples were imaged using the environmental
mode in a SEM (Quanta FEG 3D, FEI) at room temperature, 0.4–0.7 mbar,
high voltage of 20 kV, and a working distance of 3–7.5 mm.