# Quantifying Moss Response to Metal Contaminant Exposure Using Laser-Induced Fluorescence
## 4 Discussion  
The qualitative and quantitative analysis of trial 1 (control) images showed only minor variation across the three color channels for the duration of the 10-day experiment. These results would therefore suggest that the conditions maintained within the grow tent were appropriately set to sustain a stable environment for moss growth. Evaluations of images collected at day and night or before and after wetting (Figure 5) revealed that no discernable difference could be found in the control moss response over the 48 h period of examination. This gives us confidence that imaging after wetting or at different times of the day should not have affected our results. Alternatively, Cu-treated moss developed characteristic patterns discernable from qualitative observations that could be classified as shifts in the modes of red and green color distributions from higher to lower, or \"darker\" decimal code values in their scaled histograms (Figure 6). Observing that the control remained unchanged while shifts toward a taller, more right-skewed distribution occurred with increasing Cu toxicity, was qualitatively significant information. By the end of the 10-day experiment, only trial 4 exhibited discoloration in the moss observable with the human eye, but LIF results clearly show a change in fluorescence for the other trials dosed with Cu. Individual colors from the collected images were analyzed quantitatively using scaled histogram differences (Figure 9A-C) as well as a DTW technique (Figure 9D-F). Both methods allowed the difference between selected colors from trial images taken before and after Cu dosing to be represented as single numerical values. These values could then be used to compare the difference of treated trials from the mean \(\pm\) standard deviation of the control (trial 1). Analysis of the differences between scaled histograms, DTW for single-color histograms, and DTW for two-color histograms (red versus green) in Figure 10 all show treated samples imaged throughout the 10-day experiment being separated from the control mean \(\pm\) standard deviation at 3\(\sigma\) (99.7%). Agreement between all three analysis methods is encouraging, but most importantly, the results show that the LIF technique is capable of differentiating between moss treated with Cu from control samples at a Cu wet deposition level of 0.034 mg/cm\({}^{2}\) for singular doses and 0.515 mg/cm\({}^{2}\) for cumulative doses tested within this work, or potentially higher. Multi-color analysis using red vs. green color channels appeared to be more sensitive to the Cu dose, i.e., the % differences of the treated samples were larger (Tables A2 and A3). Further, when observing the results, we found that multiple incremental doses as opposed to a single dose amounting to the same Cu levels result in a comparable Cu-induced response (Figure 10). The trial 2 dose administered on day 5 (0.102 mg/cm\({}^{2}\) achieved in three doses) plots very near the trial 4 dose for day 1 (0.103 mg/cm\({}^{2}\) achieved in one dose). In comparing days 7 and 8 of trial 2 (dose of 0.138 mg/cm\({}^{2}\)) with day 3 and 4 of trial 3 (0.136 mg/cm\({}^{2}\)), the overlap can be clearly seen. These results are encouraging and suggest that LIF might be able to quantify cumulative Cu exposure in moss for both long-term environmental input and after individual deposition events. Red versus green plots of pixel counts were also explored in an attempt to better understand the co-evolution and possible relationship between changes in two colors. The produced shapes resulting from the original histograms for red and green color channels produced a looped shape, showing a shift over the experiment in which the loops both narrowed and lengthened with increasing Cu dose (Figure 8), making for a quantitatively distinct difference from the control. We were successful in employing the DTW method to quantify these shapes when compared to the control, and they separated clearly from the mean \(\pm\) standard deviation of 3\(\sigma\).