## 3 Results  
Validation of the Holomonitor M4 Digital Holographic Imaging System to Detect Norepinephrine-Induced Cardiomyocyte Hypertrophic Growth  
Chronic activation of the sympathetic nervous system can contribute to left ventricular cardiac hypertrophy [14]. Consistent with this, direct stimulation of isolated primary rat cardiomyocytes with the sympathetic neurotransmitter norepinephrine induces hypertrophy in serum-free culture [15]. We, therefore, sought to use this well-established in vitro model of cardiomyocyte hypertrophy to determine the effectiveness of the Holomonitor M4 digital holographic imaging microscope in detecting norepinephrine-dependent increases in cell volume that should accompany bona fide hypertrophic growth (Figure 1). We isolated hearts from postnatal day 1 (P1) to P3 rat pups, dissociated the tissue into single cells, cultured the cells with and without the presence of 10 uM norepinephrine, and then live imaged the cells directly on the Holomonitor M4 for 24 h (Figure 1A). Cardiac cultures from neonatal rat hearts are often contaminated with other cell types in addition to cardiomyocytes, despite methods used to increase purity [16; 17]. Our lab has recently observed that \(\alpha\)-actinin-positive cardiomyocytes can be distinguished from non-cardiomyocyte cells imaged on the Holomonitor M4 based on their overall larger size and slower motility [13] (Figure 1B). Using these criteria, we were able to readily identify cardiomyocytes in our cardiac cultures, both with and without norepinephrine treatment (Movies S1 and S2). As expected, the norepinephrine-treated cardiomyocytes exhibited significant spreading and formed cellular protrusions by the end of the 24 h treatment relative to the untreated controls (Figure 1C), which is consistent with previous observations [15]. A key technical advantage of the Holomonitor M4 digital holographic imaging system is its ability to reconstruct the three-dimensional morphologies of cultured cells. A two-dimensional increase in surface area due to cell spreading is not indicative of a three-dimensional increase in cell volume. Therefore, we utilized the HOLOMONITOR App Suite software and its Single Cell Tracking module to quantify the three-dimensional optical volumes of individual cardiomyocytes and non-cardiomyocytes at the start (0 h) and finish (24 h) of our live imaging experiments (Figure 1D,E). At 0 h, the average optical volumes of the individual untreated cardiomyocytes (\(n\) = 256) were 1380 \(\pm\) 501 um\({}^{3}\) and that of the norepinephrine-treated cardiomyocytes (\(n\) = 187) were slightly decreased at 1074 \(\pm\) 501 um\({}^{3}\) (Figure 1D). However, at 24 h, the average optical volumes of the individual untreated cardiomyocytes (\(n\) = 256) were 1341 \(\pm\) 473 um\({}^{3}\) and that of the norepinephrine-treated cardiomyocytes (\(n\) = 187) were significantly increased to 1492 \(\pm\) 501 um\({}^{3}\). These results show that during the 24 h imaging duration, the untreated cardiomyocytes did not exhibit any meaningful increase in optical volume (0.98-fold), while the volume of the norepinephrine-treated cardiomyocytes increased by approximately 1.39-fold. Non-cardiomyocytes, identified by their high motility and smaller size, did not exhibit any significant norepinephrine-dependent changes in optical volume at either the 0- or 24 h time point (Figure 1E), suggesting that stimulation with the neurohormone induced a cardiomyocyte-specific increase in cell volume. Application of the Holomonitor M4 Digital Holographic Imaging System to Monitor Real-Time Cardiomyocyte Hypertrophic Growth Dynamics  
Various technologies are capable of quantitatively evaluating cardiomyocyte size, including Coulter counter analysis [8], flow cytometry [9], and confocal imaging [10]. However, they are limited in their ability to directly and non-invasively monitor the real-time dynamics of cardiomyocyte hypertrophic responses over extended durations.