# Quantifying Moss Response to Metal Contaminant Exposure Using Laser-Induced Fluorescence
## 3 Results
### Qualitative Description of LIF Image Color Components  
#### 3.3.1 48 In Imaging Analysis  
To evaluate the moss response to the applied Cu treatment, we took images of all four trials of the moss samples at 0, 6, 12, 24, 36, and 48 h after Cu treatment. We applied the scaled histogram overlap (2) and difference (3) calculations to compare the R, G, and B color  
\begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline \multicolumn{10}{c}{**Front Cu Average and Standard Deviation in Mmol/G Dry Weight**} \\ \hline
**Day** & **1** & **2** & **3** & **4** & **5** & **6** & **7** & **8** & **9** & **10** \\ \hline
**Trial 1** & 0.24 & - & - & - & 0.32 & - & - & - & - & 0.62 \\
**Trial 2** & 20.67 \(\pm\) 10.4 & 24.95 & 23.98 \(\pm\) 19.8 & 35.07 & 62.61 & 37.12 & 197.73 & 89.69 & 128.77 & 81.42 \(\pm\) 15.5 \\
**Trial 3** & 47.79 \(\pm\) 12.8 & 27.36 & 76.69 \(\pm\) 30.4 & 43.78 & 67.71 & 84.08 & 82.82 & 69.30 & 110.08 & 100.92 \(\pm\) 39.9 \\ \hline
**Trial 4** & 70.46 \(\pm\) 13.5 & 61.99 & 169.78 \(\pm\) 13.9 & 170.45 & 339.11 & 122.05 & 211.35 & 371.17 & 301.30 & 359.62 \(\pm\) 90.9 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Cu measured in moss froms \(\mu\)mol/g dry wt. based on 3 fond pairs sampled after each treatment for each day in the experiment. Days 1, 3, and 10 show Cu averages with standard deviation for all 3 fond pairs per trial. The other days only have 1 fond pair that was analyzed so no standard deviation. Trial 1 only has data available for 1 fond pair on days 1, 5, and 10.  
Figure 4: Color histogram: number of pixels corresponding to each red, green, and blue level of intensity scaled by the total number of pixels. Top row shows images at 5 gain levels collected from the day-0 control sample using both lasers. The bottom row shows the corresponding density histogram profiles of each RGB decimal code value found in each pixel of the images. Here, (255, 0, 0) is the decimal code for red, (0, 255, 0) is the decimal code for green, and (0, 0, 255) is the decimal code for blue. Code (0, 0, 0) would be absolute black and (255, 255, 255) absolute white.  
histograms from images from all trials from 0 to 48 h to their corresponding pre-treatment (day-0) color histograms. Figure 5A shows the red channel results (though all colors show the same trend) for the control (trial 1; no Cu treatment) versus the other trials, which were dosed with Cu. The control images taken during day (light) versus night (dark) and pre- versus post-wetting after 24 h do not show any significant difference. Cu-dosed trials, however, showed a non-linear trend in moss response over time corresponding to initial Cu treatments. The three moss samples that received three different levels of Cu doses had very distinct responses as measured by their LIF images. Each of the treated samples showed an increasing deviation of the red histogram from the pre-treatment day-0 control following a Cu dose, but responses began to flatten after 12-24 h. Red histogram differences were also used to represent each trial based on the Cu dose received (Figure 5B) and showed increased differences with increasing dose. Figure 5B also shows the variability of results over the 48 h time period after a given Cu dose was applied. While this work did not study the underlying physiological changes in moss after adding Cu, our results suggest that a change in LIF response can be observed for at least 24-48 h after treatment. Accordingly, the experiment was set to apply Cu doses every 48 h and produce LIF images every 24 h throughout the 10-day duration of the experiment.