Visualizing Melanin in Lichen Thalli

To image melanin of L. pulmonaria by scanning and transmission electron microscopy, thalli were embedded in 3% agarose blocks and cut using a vibratome (Leica VT 1000S, Wetzlar, Germany), resulting in 50 µm cross sections from the upper cortex. The cross sections of pale and melanized lichen thalli were subsequently fixed in 2.5% glutaraldehyde in 0.1 M Na phosphate buffer, pH 7.4, and 1% osmium tetroxide, and further dehydrated as described by Daminova et al. (2022) [10 (link)]. Sections were then sputter-coated with gold using the Q150T ES Coater (Quorum Technologies, Lewes, UK), and anticlinal and periclinal cell walls were viewed using a high-resolution scanning electron microscope (Merlin, Carl Zeiss, Oberkochen, Germany) at a voltage of 5 kV.