## 3 Results  
### Monitoring of Epithelial to Mesenchymal Transition Using DHC  
Digital holography is a non-invasive imaging technique that provides three-dimensional morphological data of cells in a monolayer culture. Since the technique is non-invasive and label free, it is suitable for long term live imaging. We routinely use this technique to image adherent mammalian cells at regular intervals for 24-72 h. The individual images from each timepoint are then segmented and DHC-derived morphological features are calculated, so that changes in these features over time can be monitored. To first determine whether DHC can distinguish between extreme epithelial and mesenchymal states of the same line in a well-defined system, we cultured the mouse mammary epithelial cell line (NMuMG) in the presence or absence of TGF\(\beta\), an established potent inducer of EMT in this context [27; 28; 29]. This EMT can be inhibited by co-treatment with TGF\(\beta\) type-1 receptor blocker [27; 28; 29]. The cells were treated with 0.2 or 0.5 ng/mL of TGF\(\beta\) for a total time of 72 h to induce the transition of the epithelial NMuMG cells to mesenchymal cells. Figure 1e,f show that the morphology of cells treated with TGF\(\beta\) is different compared to control (Figure 1a). Specifically, control cells present typical epithelial morphology defined by uniform round cells growing in tight clusters often described as having a \"cobblestone\" appearance. In contrast, the TGF\(\beta\)-treated cells were elongated, and clusters were less compact with individual cells migrating away, which are characteristics associated with the mesenchymal state. Cells treated with the TGF\(\beta\) type-1 receptor inhibitor SB431542 [28] added together with TGF\(\beta\) (Figure 1d) are similar to control (Figure 1a). Treatment with the inhibitor alone at either 0.5 uM or 1 uM concentration had no effect on cell morphology (Figure 1b,c), however the higher concentration reduced the cell number. Cells were identified in DHC images generated at 0, 24, 48, and 72 h and 27 DHC-derived features were presented by Hstudio as output parameters. To determine which parameters demonstrated a significant correlation with the EMP spectrum, the treatments were ordered and assigned a numerical value in relation to increased mesenchymal features according to our own observations and published results [27; 28; 29]. The non-treated control was set as 1; treatment with 0.5 uM TGF\(\beta\)RI was set as 2; treatment with 1 uM TGF\(\beta\)RI was set as 3; treatment with the combination of 0.5 ng/mL TGF\(\beta\) and 0.5 uM TGF\(\beta\)RI was set as 4; treatment with 0.2 ng/mL TGF\(\beta\) was as 5; and treatment with 0.5 ng/mL TGF\(\beta\) was set as 6. We then calculated the correlation coefficient between each parameter and the EMP numerical assignment (Figure 2a). At time-point 0, no correlations were observed, indicating that immediately after treatment the cells were morphologically identical regardless of the condition. However, as the treatment time increased, increasing correlations between several features and treatment condition were identified, suggesting a consistent morphological change over time. To determine which features provided redundant information in this context, we calculated the correlation coefficient between each pair of features (Figure 2b). We identified four groups of features with near perfect correlation. In order to restrict further analyses to independent parameters, one feature from each group that was correlated to the conditions was selected (Figure 2a, green boxes). These features were cell eccentricity (a measure of cell roundness), hull convexity (a measure of how dome-shaped the cell is), optical thickness max (a measure of cell thickness or optical density), and roughness skewness (a measure of cell texture). Figure 1: Treatment of NMuMG cells with TGF\(\beta\) induces EMT which is inhibited by the TGF\(\beta\) type-1 receptor inhibitor SB431542.