## 3 Results
### Changes in the Onion Epidermal Cells in Response to PEF  
Representative cryo-SEM micrographs of untreated and PEF-treated onion tissues (Figure 4) showed that there was a considerable difference in the onion epidermal cells in response to PEF. Figure 4A shows the adaxial (inner) surface of fleshy scale 2 from an untreated onion bulb that was damaged during sample preparation (with the tunic layer removed), thus revealing intact and turgid underlying storage circular cells. Likewise, Figure 4B shows minimal damage on the abaxial (outer) surface of fleshy scale 2 from an untreated onion bulb, showing intact and turgid elongated epidermal cells beneath the cuticle layer. Abaxial epidermal cells had dimensions of approximately \(250.44\pm 16.39\)\(\upmu\)m \(\times\)\(67.48\pm 3.80\)\(\upmu\)m (length \(\times\) width) (\(n=10\)); while, the relative isometric storage circular cell had a mean diameter of \(204.20\pm 8.37\)\(\upmu\)m (\(n=10\)). The effect of PEF applied at 1.2 kV/cm (PEF 3) with the NFE orientation on the adaxial surface of a PEF-treated onion was observed at the fleshy scale 3 (Figure 4C). The underlying storage circular cells had lost their turgidity and many cells were collapsed (Figure 4C), in comparison to the storage circular cells in the untreated onion scale 3 (Figure 4D), where the storage cells at the cut edge of the specimen were mostly intact and turgid. Some minor surface damage can be seen because of tunic layer removal, as shown in Figure 4D with an arrow.  
Severe cell damage caused by a \"PEF 3\" treatment with an onion orientation where its root end was facing the high voltage electrode (FE) can be seen on the adaxial surface of scale 2, as illustrated in Figure 4E. The observed flat surface was made up of dead cells that completely lost their cell wall integrity. On the abaxial surface of scale 2 obtained after a \"FE-PEF3\" treatment (Figure 4F), there was minimal surface damage, which was similar to the untreated scale 2 evaluated at the abaxial surface (Figure 4B). However, the severe damage on the storage circular cells (Figure 4E) caused a reduction in tissue thickness and density due to the loss of cytosol and cellular turgidity leading to wrinkle formation on the cuticle layer (Figure 4F; arrow).  
Figure 4G,H shows the effect of a specific energy input of 60 kJ/kg (PEF 2) with the FE orientation on the adaxial and abaxial surfaces of a PEF-treated onion observed in fleshy scale 3. Figure 4G specifically shows that \"FE-PEF2\" caused a complete disruption of the adaxial cell layers and tissue structure similar to Figure 4E. In contrast, \"FE-PEF2\" caused minimal damage on the cuticle layer with the epidermal cells mostly intact and turgid (Figure 4H). It was also found that the epidermal cells became smaller and less elongated in the adaxial scales compared to the other abaxial scales in response to \"FE-PEF2\" treatment.  
Overall, the results from cryo-SEM showed that PEF had minimal damage on the abaxial surface, whereas most damage was found on the adaxial surface of the fleshy scales. Abaxial epidermal onion cells retained their cell wall integrity following PEF treatment (Figure 4F,H); while the adaxial storage circular cells lost their cell wall integrity (Figure 4C,E,G).  
Figure 3: Representative photographs of untreated and PEF-treated onion bulbs over the course of 6 h after PEF application. The onion bulb was positioned with either the root end facing the electrode (FE) or the root end not facing the electrodes (NFE) at the time of PEF application, then suspended in the sodium phosphate buffer (0.01 M, pH 7) and removed at a predetermined time point to capture the changes in the natural purplish-red pigment distribution.