# Polarimetric Detection of Chemotherapy-Induced Cancer Cell Death
## 4 Results and Discussion
### Cell State Assessment: Quantifying Cell Death  
U937 leukemia cells were used to perform the polarimetric study related to the effect of a chemotherapy treatment. Cells were treated with the cytotoxic drug cisplatin. Viability tests were performed at 24, 48, and 72 h after treatment. In addition, polarimetric measurements of samples deposited on a microscope slide were carried out. Control (non-treated) and treated cells were compared. Ordinary microscopic images (Figure 4) showed the differences between untreated (A) and the cisplatin-treated cells at 24 (B), 48 (C), and 72 (D) hours. Cisplatin-treated U937 cells were less confluent, showed cell shrinkage, and became pyknotic (with cytoplasmic and nuclear condensation) at longer times, mostly for 72 h (Figure 4D). These morphological features are associated with apoptotic cell death [40], and it is well established that cisplatin induces apoptosis in a number of cancer cells, including U937 cells [41]. We performed the trypan blue assay to evaluate cell viability at different time points of treatment (Figure 5). Cell mortality gradually increased over time, reaching about 80% cell death by 72 h. This result was confirmed by showing that cisplatin reduced the metabolic activity of U937 cells from 2.4-fold at 24 h to 100-fold at 72 h, corresponding to cells undergoing cell death (Figure 6). Figure 4: U937 sample images taken by phase contrast microscopy. (**A**) Control sample (non-treated); (**B–D**) 24, 48, and 72 h after treatment with 20 \(\upmu\)g/mL of cisplatin. The white dashed square represents the size of the image taken with the polarimeter (640 \(\times\) 640 pixels. )After that, five images (640 \(\times\) 640 pixels) of the Mueller matrix were measured in five different regions of each of the four samples. The five different regions were randomly selected in the area covered by the cells (circle of 6 mm in diameter). The dashed white square in Figure 4D) represents the size of the image taken with the polarimeter (180 \(\times\) 180 \(\upmu\)m approximately). Some polarimetric magnitudes were obtained by applying different decomposition methods, and finally, in order to analyze the results, the mean values of each magnitudes were calculated. Figure 7a shows the mean value of the five images of diattenuation for each sample. A slight, but significant increase was observed at 24 h (\(p\)-values obtained with both tests were 0.016), when cell death was about 25%. Great differences were observed by 48 h of treatment, when the cell death was above 50%. As was explained in Section 3, we found that the results were highly dependent on cell confluence, this is the filling factor \(f_{c}\) of the image. In Figure 7, both the raw results and those corrected according to Equation (12) are represented. Then, we used two statistical non-parametric tests to check if it was possible to tell apart one measurement set from another. We applied the Wilcoxon rank test and the Kruskal-Wallis test to our set of data and obtained the corresponding \(p\)-values (Table 1). Figure 5: Cell death of U937 cells over time treated with 20 \(\upmu\)g/mL of cisplatin evaluated with the trypan blue exclusion test. The error bar is the standard deviation of three measurements. Figure 6: Metabolic activity of U937 cells following a treatment with cisplatin at different time points using the Alamar Blue assay. a.u., arbitrary units. The error bar is the standard deviation of three measurements. Statistical analysis tells us that there was a significant difference between the population of untreated (control) and treated cells (\(p\)-values < 0.05). This confirms that we were able to detect even low percentages of cell death. At longer times, the correlation between cell death and diattenuation became stronger with \(p\)-values smaller than 0.01. Other polarimetric magnitudes obtained from different decomposition methods showed a similar variation over time.