## 3 Results
### Effect of the Generated (Reactive) CAP Species on the Biofilms
#### 3.2.4 Confocal Laser Scanning Microscopy (CLSM)  
Figure 9 provides an overview of the CLSM images obtained following CAP treatment of the two model biofilms using each possible combination of plasma characteristics. 2D-images at different depths of the biofilms were provided to examine the ability of the generated CAP species to penetrate into the deeper layers of the model biofilms (from left to right: top layers to deeper layers). Moreover, these 2D-images can be used as well to (i) comment on possible changes in the integrity of the cell membranes and (ii) examine if the CAP treatment has an effect on the biofilm structure.  
Figure 8: Fluorescence measurements (-) obtained following interaction of SYBR Green I with the CAP treated and digested _Listeria monocytogenes_ and _Salmonella_ Typhimurium-associated cells (\(n=3\)). Significant differences between all examined conditions (ANOVA test 1), including the untreated control, have been indicated by means of a different small letter, with ‘a’ bearing the lowest value. The influence of the oxygen level (ANOVA tests 2–5), the electrode configuration (ANOVA tests 6–9), and the discharge power (ANOVA tests 10–13) have been indicated as well by means of a different small letter, with ‘a’ again bearing the lowest value. Separate ANOVA tests were again performed for each biofilm forming species.  
Figure 9: _Cont._  
Figure 9: Two-dimensional CLSM images taken at different depths of the (CAP treated) model biofilms, with images from left to right going from the top to the bottom layers of the biofilm (\(n=1\)). Images have been provided for both biofilm forming species and for each examined combination of plasma characteristics (i.e., DBD/SBD, He + 01% (v/v) oxygen, and 13.8/21.88 V input voltage). CAP treated and control biofilms were always stained with a mixture of calcofluor white (EPS, blue emission), SYTO9 (living cells, green emission), and propidium iodide (dead/damaged cells, red emission) prior to the visualization step. Depending on the viability of the biofilm-associated cells and the presence of EPS components within a specific biofilm layer, this resulted in two-dimensional images with different staining patterns.  
First of all, it is important to mention that the untreated control biofilms already contained a significant amount of dead/damaged (red) cells. This has been observed before in the study of [PERSON] et al. [26] and this was deemed to be the consequence of the applied biofilm formation conditions resulting in (sub-lethal) injury of the cells. The intensity of the dead/damaged (red) cells, however, increases following CAP treatment of the model biofilms, indicating that the number of viable cells can be significantly reduced following CAP treatment and/or that this treatment was able to cause damage to the membrane of the cells. Moreover, for all examined CAP treatment conditions, cell damage/injury was observed into the deeper layers of the biofilms. As a result, it can be concluded that the inhibition of CAP species to penetrate into the model biofilms is rather limited. Secondly, regarding the influence of the plasma characteristics on the ability of the CAP species to penetrate into the model biofilms, no major conclusions can be drawn based on the obtained CLSM images since cell death/damage was observed in each layer of the biofilms and for each combination of plasma characteristics. Further research will be required to analyze this in detail, e.g., by means of diffusion measurements. Finally, it appears that some treatment conditions resulted in the formation of holes into the _Listeria monocytogenes_ biofilms, indicating that the matrix of this model biofilm can be damaged by means of CAP treatment and/or that the cells show some kind of motility reaction upon exposure to CAP.