# Polarimetric Detection of Chemotherapy-Induced Cancer Cell Death
## 4 Results and Discussion
### Cell State Assessment: Quantifying Cell Death
We considered that values of the depolarization index corresponding to the samples were too small and close to the values obtained in the calibration (Figure 2a) to be significant. Figure 8 shows the evolution of the \(m_{11}\) element (intensity image) and five polarimetric magnitudes (total diattenuation, anisotropy, total dichroism, total birefringence, and depolarization index) over time before and after applying the filling factor correction. Bars represent the mean values of all the pixels of the five images taken in each sample. The intensity image (\(m_{11}\)) was not useful for extracting conclusions because was a non-normalized parameter, and it depended on the illumination \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**TEST** & **C-24** & **C-48** & **C-72** & **24–72** & **48–72** & **24–72** \\ \hline W. rank sum & 0.016 & 0.008 & 0.008 & 0.008 & 0.008 & 0.008 \\ Kruskal-Wallis & 0.016 & 0.009 & 0.009 & 0.009 & 0.009 & 0.009 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: \(p\)-values for each possible couple of sets of measurements of the first experiment (C is the Control) after the filling factor correction. Values with \(p\) < 0.01 mean a high significance, values between 0.01 < \(p\) < 0.05 significant, and values of \(p\) > 0.1 not significant. Figure 7: Mean diattenuation (\(D_{T}\)) of each of the five images taken from the control and cisplatin-treated samples of U937 cells. (**a**) shows the five measurements taken for each time in the first experiment. (**b**) shows the mean values at each time for the triplicate experiment (numbered as 1, 2, and 3). Data are shown before (\(\square\), dashed bars) and after (\(\bullet\), solid line) applying the the correction given by Equation (12). When the bar is not duplicated, it means that \(f_{c}\) = 1 for the measured image, and no filling correction is required. Error bars represent the standard deviation of the image. and on the integration time of the camera. However, all five magnitudes seemed to be sensitive to the cell death and presented a similar trend over time. We decided to focus our study on diattenuation because it presented the lowest standard deviation. Once focused on diattenuation, we performed our experiment in triplicate. Results are shown in Figure 7b, where the mean values of diattenuation are presented for each experiment and for each time point. In Figure 9, we show the correlation between the mean values of diattenuation and the mean value of the percentage of dead cells obtained from the triplicate experiment with U937 cells. Both diattenuation and cell death increased over time. At 24 h, there was a slight increase in diattenuation that became very significant at 48 h of treatment. If data were not corrected by the filling factor, by 72 h, the diattenuation slightly decreased with respect to the previous time point, likely due to the loss of cell confluency, as shown in Figure 4D, which would decrease the mean value of the parameter measured. After correction we see that the diattenuation increased with the cell death. Figure 8: Mean total diattenuation, anisotropy, total birefringence, total dichroism, and depolarization index of the five measurements made at each time in the first experiment. Data are shown before (\(\square\), dashed bars) and after (\(\bullet\), solid line) applying the correction given by Equation (12). When the bar is not duplicated, it means that \(f_{c}=1\) for the measured image. Error bars represent the mean standard deviation of the five images of each parameter. A similar result was obtained when another leukemia cell line, THP1, was treated with cisplatin. By 24 h of treatment, there was over a two-fold increase of cell death by trypan blue (Figure 10a), which was confirmed by a reduction of metabolic activity (Figure 10b). Consistent with our previous data, the diattenuation significantly increased (\(p=0.008\) with the Wilcoxon rank test) after treatment with cisplatin for 24 h (Figure 11a)). Results for the triplicate experiment are shown in Figure 11b).