Gatan alto 2500

A Hitachi S-4800 (Hitachi High-Technologies Corp., Tokyo, Japan) cryo-SEM equipped with a Gatan ALTO 2500 cryopreparation system (Gatan, Inc., Abingdon, United Kingdom) was used for imaging the flower surface details. Therefore, the viscidium of a fresh flower was partly removed manually using the head of a pin and mounted on metal holders with Tissue-Tek ® O.C.T. ™ Compound (Sakura Finetek Europe B. V., Zoeterwoude, The Netherlands). The samples on holders were frozen on a cryostage at 140 °C, sputter-coated with gold-palladium (6 nm) in the cryopreparation chamber, and examined in the SEM at -120 °C and an accelerating voltage of 1 kV.

The Greek style full fat and non-fat yoghurts were analysed by cryo scanning electron microscopy (cryo-SEM) following a previously published method [37] (link). Yoghurt microstructure was observed using a scanning electron microscope (SEM; Zeiss Supra 40VP field emission, Carl Zeiss AG, Darmstadt, Germany) with a cryo sample preparation system (Gatan Alto 2500, Gatan UK). Fresh samples were rapidly immersed in liquid nitrogen slush (-210°C) and transferred under vacuum to the cryo-preparation chamber maintained at -140°C. The samples were fractured with a scalpel blade and followed by etching at -95°C. Sputter coating was conducted at -140°C and the sample transferred to the SEM cold stage maintained at -140°C. Cryo-SEM micrographs were collected over a range of magnifications with one representative image shown per sample. The yoghurt microstructure observed by cryo-SEM was used as a comparison to that observed using confocal Raman microscopy.

The remaining three root segments of E. nocturnum (with attached substrate) were prepared for cryo-SEM examination. The samples were shock-frozen (-140 • C) to study the rootsubstrate interface in a SEM Hitachi S-4800 (Hitachi High-Technologies Corp., Tokyo, Japan) equipped with a Gatan ALTO 2500 cryo-preparation system (Gatan Inc., Abingdon, United Kingdom). For details of sample preparation and mounting for cryo-SEM, see Gorb and Gorb (2009) . Cryo-SEM is useful, where preservation of the natural morphology of cells and tissues is desired. Additionally, it allows visualization of fluids, which might be crucial in the case of root-substrate attachment. Mounted root segments with bits of bark substrate attached were sputter-coated in frozen conditions with gold-palladium (thickness 10 nm) and examined at 3 kV acceleration voltage and temperature of -120 • C in the microscope.
To ensure that the results of the cryo-SEM analyses were not artifacts due to the preparative procedures and long storage in ethanol, three 1 cm fresh root samples of another epiphytic orchid, Dendrobium koordersii J.J.Sm, mounted onto Robinia pseudoacacia bark, were collected in January 2021 in the greenhouse in the Botanical Garden Kiel, Germany. All plant names follow The Plant List (2013).