## 4 Discussion  
Using advanced microscopical analysis, the present study provides an accurate morphological characterization of _U. barbata_, offering a complete structural investigation. The  
Figure 7: The Principal Components Analysis (PCA) plot. microstructure of _U. barbata_ integral and fractions displayed in our study agreed with those reported by [PERSON] et al. [37] and [PERSON] et al. [21]. The whole lichen consists of 90% mycobiont and only 10% photobiont [24]. Of the separated fractions, one is formed exclusively of mycelial hyphae (_cc_UB), and the other (_mc_UB) has both symbionts. It is known that, in symbiotic partnerships, only the fungus synthesizes the lichen's secondary metabolites. Thus, we wanted to know which lichen fraction had phenolic metabolites and a mineral content higher than the entire lichen. Being a source of bioactive metabolites, extracting them from the fraction with the highest content could increase the process yield and selectivity. [PERSON] et al. [21] revealed an entire micrograph of a cross-section of _Usnea_ sp. thallus obtained by SEM-EDS analysis. These authors separated only two _U. barbata_ thallus fractions (cortex-medulla and axis), similar to those described in our study (_mc_UB and _cc_UB). The cortex consisted of densely packed thick-walled fungal hyphae in their image, and the central cord had a compact, cartilaginous structure [21]. Most numerous organic compounds found in lichens are secondary metabolites of fungal origin [20]. Their amount is usually 0.1-10% in dried lichen and can reach 30% in some cases. These substances are mostly phenolic compounds, poorly soluble in water, and can be extracted using organic solvents [42]. They are deposited in the cortex and medulla as crystals on the hyphae surface. The lichen metabolites are differentially distributed in the thallus layers, correlated with their bioactivities [20]. Thus, the compounds from the upper cortex act as a light filter, having a photoprotective effect [40]. The most common cortical metabolites are usnic acid, atranorin, xanthones, pulvinic acid derivates, and anthraquinones. With few exceptions (atranorin), they are colored compounds (yellow, orange), giving the lichen thalli specific coloration [40]. These cortical constituents can be distinguished from numerous medullary ones [20]. Bioactive metabolites localization in the lichen thallus was explored using various methods. Some researchers used spot tests (directly applying suitable reagents to the lichen thallus) or after extraction and analyses of specific lichen parts [43]. Other studies described classical fluorescent techniques based on the molecule chromophore, where metabolites are only differentiated by their emission wavelength; then, the metabolite localization in living plant cells was obtained using multiphoton microscopy [44]. In 2016, based on the auto-ionisability of all main classes of lichen bioactive compounds [45], [PERSON] et al. [46] achieved histolocalisation of the lichen _Opiiaparma ventosa_ by laser desorption ionization coupled to mass spectrometry imaging (LDI-MSI). They applied different slicing procedures to obtain lichen thalli transverse sections (cryosectioning and hand-cutting), fixed them on a carbon-conductive adhesive tape, and examined the metabolites' spatial distribution. MSI displayed the molecular images of usnic acid, thamonic acid, divaratic acid, miriquidic acid, haemoventosin, and all overlaid ions in cryosectioned pieces of the _O. ventosa_ thallus [46]. Using the same method, [PERSON] et al. [43] obtained spatial mapping that revealed phenolic metabolites in the lichen _Pseudocympellaria croxata_. Moreover, for optimal extraction of localized lichen metabolites, different methods of sample preparation were elaborated. [PERSON] et al. [47] proposed two types of preparations for atranorin extraction from _Pseudevernia furfuracea_. In the first case, the lichen sample was milled (with a planetary ball mill), resulting in a homogeneous powder.