## 3 Results
A major advantage of the Holomonitor M4 is that it utilizes a low-power 635 nm laser, which minimizes phototoxicity, making the instrument ideal for long-duration live-cell imaging [12]. Cardiomyocyte size is typically evaluated by an increase in two-dimensional surface area or three-dimensional volume. Therefore, we questioned if the Holomonitor M4 could be uniquely applied to quantitatively track dynamic changes in cardiomyocyte surface area and optical volume throughout our 24 h imaging experiments (Figure 2A). The HOLOMONITOR App Suite analysis software allows for in-depth quantitative analysis of cell morphology and behavior. We used the Single Cell Tracking module to apply image thresholds to segment each frame of our time-lapse videos and identify individual cardiomyocytes (Figure 2B). We then manually selected well-isolated cardiomyocytes to follow and track throughout the entire 24 h imaging duration. We verified the accuracy of segmentation for each tracked cell in each imaging frame. This was assisted by the HOLOMONITOR App Suite software, which automatically flagged cells that exhibited dramatic changes in cell volume for closer inspection (Figure 2B). Cells that converged with other cells or those for which tracking became ambiguous at any point during the imaging period were omitted from analysis. We first analyzed the surface area dynamics of both the untreated and norepinephrine-stimulated cardiomyocytes over the 24 h imaging period (Figure 2C). T-test comparisons were applied at each time point to determine instances when surface areas statistically differed between the two experimental conditions. This analysis suggested that norepinephrine increases cardiomyocyte surface area as early as 10 h after treatment. However, it is important to note that the untreated cardiomyocytes also exhibited a mild norepinephrine-independent 1.2-fold increase in surface area by the end of the imaging experiment. This could be due to the presence of cardiac fibroblasts in culture, which have been reported to increase cardiomyocyte surface area in vitro [18]. Therefore, it may be difficult to uncouple norepinephrine-dependent and independent effects on cardiomyocyte hypertrophy when measuring only surface area. When we analyzed cardiomyocyte three-dimensional optical volume dynamics, we noticed that the untreated cells cultured in our serum-free conditions exhibited little change in their average optical volume over the entire imaging duration (0.98-fold), suggesting that optical volume may behave more stably in our experimental timeframe. However, the average starting optical volumes of the norepinephrine-treated cardiomyocytes were significantly decreased compared to that in the untreated control cells (Figures 1D and 2D). Therefore, we could not reliably evaluate when norepinephrine stimulation induced a statistically significant change in cardiomyocyte volume due to these differences in baseline measurements. The Holomonitor M4 and HOLOMONITOR App Suite software permit longitudinal tracking of cardiomyocyte dynamics with single-cell resolution. This enabled us to normalize the size of each individual cardiomyocyte based on its initial value measured at the start of our imaging experiments (Figure 2E,F). This analysis revealed a clearer dynamic of norepinephrine-stimulated cardiomyocyte hypertrophy. T-test comparisons applied at each time point revealed statistically significant norepinephrine-dependent increases in optical volume as early as 2 h after the start of our imaging experiments (Figure 2F). Taken together, these results provide proof-of-principle evidence that the Holomonitor M4 digital holographic imaging system can be successfully applied to monitor dynamic changes in cardiomyocytes surface area and volume in response to a hypertrophic stimulus.