## 4 Discussion  
In this study, we evaluated the application of a commercially available Holomonitor M4 digital holographic imaging microscope to detect dynamic changes in primary neonatal rat cardiomyocyte surface areas and optical volumes in response to norepinephrine stimulation leading to hypertrophic growth. The Holomonitor M4 utilizes a low-powered 635 nm laser to generate holograms for three-dimensional image reconstruction [19], promoting live-cell imaging over extended durations [20]. However, if phototoxicity is observed, it may be possible to acquire time-lapse images at longer time intervals to further minimize the risk. This label-free imaging approach has great potential to characterize the cellular dynamics underlying cardiomyocyte responses to hypertrophic stimuli and also other cardiomyocytes, including adult and iPS-derived cardiomyocytes [21]. Our results provide evidence that this imaging system can reliably detect dynamic increases in cardiomyocyte surface area and volume that accompany the hypertrophic response to the model stimulus, norepinephrine (Figure 2C-F). This has significant potential to enable future studies to explore the temporal aspects of cardiomyocyte hypertrophic responses. Additionally, these studies could further exploit the advantages of this approach to characterize cardiomyocyte responses to various hypertrophic inducers and investigate potential synergies between them. However, our results also shed light on the technological limitations of the approach that should be considered. Single-cell tracking using the HOLOMONITOR App Suite software revealed a statistically significant decrease in the starting optical volumes of the norepinephrine-stimulated cardiomyocytes(Figures 1D and 2D). It is unlikely that the cardiomyocyte volumes truly differed between the untreated and treated cells at the start of the experiment since the cardiac cells in both conditions were derived from the same tissue preparations. While we attempted to start our digital holographic imaging time-course experiments soon after norepinephrine treatment, the time it took to initiate our time-lapse movies after treatment ranged from 1 to 2 h due to technical obstacles. These include the time it takes to reacclimatize cultures back to 37 \({}^{\circ}\)C to resolve condensation on the culture plate imaging surface, which interferes with image acquisition. Additional time is also spent operating the analysis software and identifying adequate imaging fields prior to starting time-lapse acquisition. Our results show that norepinephrine has a strong cardiomyocyte cell spreading effect by 24 h (Figure 1C). Therefore, it is possible that cardiomyocytes may have already started to spread soon after norepinephrine stimulation during our 1-2 h transition time prior to image acquisition. This would likely impact the fidelity of image segmentation and cardiomyocyte identification since it is more difficult for the analysis software to distinguish signal from background noise in thinner cellular processes than in thicker regions of the cell (Figure S1). Compared to cells exhibiting a more compact and rounded morphology, this error would be magnified in cardiomyocytes that strongly spread over the substratum, which would lead to an underestimation of volume measurements. Therefore, cell spreading could be a confounding factor when interpreting raw unnormalized morphology measurements observed between control and treatment conditions and should be accounted for in the experimental design. This cautions against the interpretation of raw morphological measurements as absolute since the accuracy of these measurements is dependent on segmentation accuracy and cell thickness (Figure 2C,D). Nevertheless, the approach may be useful for gaining insights into the relative dynamics of underlying hypertrophic processes (Figure 2E,F). Another limitation of the technology is that accurate image segmentation and the identification of individual cardiomyocytes requires well-isolated cells that are not integrated into clusters.