## 3 Results and Discussion
### Investigations on the Effect of Ultrasound on the Structure of an Olive Leaf
#### 3.2.2 Histological Structure of the Leaf  
To establish a link between polyphenol extraction enhancement by ultrasound and modifications noticed on the leaf surface, a histology study was conducted. Leaf sections were stained with toluidine blue O (TBO) to localize phenolic compounds. For homogeneity, the leaves from the same experimental conditions as ESEM were compared in Figure 3.  
For the control leaf, TBO stained the hair as well as the cuticle (Figure 3A). As shown in ESEM observations, the abaxial surface was covered with hairs (Figure 3B). From the coloration intensity distribution with TBO, we concluded that the feet of hairs were particularly rich in phenolics. Flavonoids have been identified in hairs and in the epicticular wax of olive leaves [33; 36]. The authors also showed that in mature olive leaves, phenolic compounds tend to be concentrated in the wall of cells composing the hairs [33], converging towards the hypothesis of a protective action of hairs against UV. Both parenchymas (palisadic and spongy) were also strongly stained with TBO (Figure 3A,B). The structure of the control leaf (Figure 3C,D) was identical to the one of the untreated leaf. Figure 2: Comparison of leaf surface by Environmental Scanning Electron Microscopy (ESEM) for an untreated leaf, a control leaf (60 min stirring), and ultrasound treated leaves (5 min, 15 min, and 60 min). Legend (Figure 2): Cuticle (C), Hairs (H), and Dust Particles (DP). All observations were made at a magnification of 400\(\times\) the original picture’s scale and the scale is 200 \(\upmu\)m. Non labeled arrows designate observations of leaf structures discussed in the Results section. (**A,B**) Untreated olive leaf abaxial and adaxial side, respectively. (**C,D**) Control leaf, after 60 min of stirring at 20 \({}^{\circ}\)C, abaxial and adaxial side, respectively. (**E,F**) Leaf treated with ultrasound for 5 min at 20 \({}^{\circ}\)C, abaxial and adaxial side, respectively. (**G,H**) Leaf treated with ultrasound for 15 min at 20 \({}^{\circ}\)C, abaxial and adaxial side, respectively. (**I,J**) Leaf treated with ultrasound for 60 min at 20 \({}^{\circ}\)C, abaxial and adaxial side, respectively. The histology of ultrasound-treated leaves showed modifications located at the surface of the leaves. A 5 min ultrasound duration seemed to impact the adaxial surface with hair removal (Figure 3E); however, on the abaxial surface, the hair coverage was still high (Figure 3F). After 15 min of sonication, modifications of hair structure occurred. On the adaxial surface, hairs seemed to be dissociated (Figure 3G) or otherwise totally removed (Figure 3I). Additionally, the cuticle thickness seemed to be reduced on the leaf sonicated  
Figure 3: Comparison of olive leaf cross sections tainted in toluidine blue by optical microscopy for untreated leaf, control leaf (60 min stirring), and ultrasound-treated leaves (5 min, 15 min, and 60 min). Legend (Figure 3): Cuticle (C), Epidermis (E), Hair (H), Palisadic Parenchyma (PP), Lacunary Parenchyma (LP), Hair Layer (HC). All observations were made at a magnification of 20\(\times\) the original picture’s scale. Bars: 100 \(\upmu\)m. Non labeled arrows designate observations of leaf structures discussed in the result section. (**A,B**) Untreated olive leaf abaxial and adaxial side, respectively. (**C,D**) Control leaf, after 60 min of stirring at 20 \({}^{\circ}\)C, abaxial and adaxial side, respectively. (**E,F**) Leaf treated with ultrasound for 5 min at 20 \({}^{\circ}\)C, abaxial and adaxial side, respectively. (**G,H**) Leaf treated with ultrasound for 15 min at 20 \({}^{\circ}\)C, abaxial and adaxial side, respectively. (**I,J**) Leaf treated with ultrasound for 60 min at 20 \({}^{\circ}\)C, abaxial and adaxial side, respectively. for 60 min (Figure 3I). On the abaxial surface, for both 15 min and 60 min of sonication, hairs seem to be emptied, destructured, and less numerous (Figure 3H,J).