## 1 Introduction  
Cardiomyocyte hypertrophy--an increase in the size of individual heart muscle cells--is a hallmark feature of many cardiovascular diseases that may arise from a number of pathological conditions including hypertension, myocardial infarction, and valve dysfunction. While initially considered an adaptive response to hemodynamic stress or injury, sustained myocardial hypertrophic growth often progresses to heart failure, a leading cause of morbidity and mortality worldwide [1]. Pathological hypertrophy is often accompanied by adverse structural and functional alterations in heart function due to fibrosis, impaired contractility, and arrhythmogenesis [2]. Pathological hypertrophy often precedes the development of heart failure and other adverse cardiovascular events [3]. Understanding the dynamics of pathological cardiac hypertrophy at the cellular level may lead to the development of novel treatment strategies to mitigate disease progression and improve patient outcomes.  
While in vivo studies have provided invaluable insights into the physiological signals underlying pathological hypertrophy of the heart, in vitro primary cardiomyocyte culture models have contributed significantly to our understanding of the cellular and molecular mechanisms that drive cardiomyocyte hypertrophic responses. For instance, studies have employed primary cardiomyocyte cultures to explore the effects of various hypertrophic stimuli, such as mechanical stretch, neurohormonal factors, and growth factors [4; 5]. Additional studies have revealed key intracellular signaling cascades, including the calcineurin-NFAT and MAPK pathways, that contribute to hypertrophic growth responses [6; 7]. Overall, in vitro primary cardiomyocyte culture models have served as valuable tools for dissecting the multifaceted processes that drive cardiomyocyte hypertrophic growth and greatly complement in vivo studies. However, despite the advantages of in vitro primary cardiomyocyte culture models, the current strategies used to assess cardiomyocyte size and hypertrophic growth are limited to static end-point measurements [8; 9; 10]. Surface area assessments of cardiomyocytes in two dimensions [11] overlook alterations in cell thickness and primarily focus on cell spreading. Coulter counters [8] and flow cytometry [9] offer measurements at the population level but lack the ability to capture individual cell dynamics. Additionally, three-dimensional confocal imaging [10] often requires cell fixation and optical sectioning using high-intensity lasers, thereby limiting the prolonged observation of live cardiomyocytes. Therefore, the temporal dynamics of cardiomyocyte hypertrophic responses have remained highly understudied.  
The Holomonitor M4, a commercially available digital holographic imaging microscope small enough to fit within standard cell culture incubators, permits the non-invasive, label-free, long-term quantitative three-dimensional imaging of individual living cells [12]. An interference pattern created between a sample and a reference beam generates phase shift data, which are then reconstructed to visualize the cultured cells in three dimensions. The goal of this study is to evaluate if the Holomonitor M4 imaging system can be applied to quantitatively track the three-dimensional dynamics of cardiomyocyte hypertrophic responses in real-time. The powerful Holomonitor App Suite analysis software facilitates quantitatively monitoring single-cell behaviors and morphology simultaneously. We aim to determine if these unique imaging and tracking capabilities can be leveraged to provide further insights into the temporal aspects of cardiomyocyte hypertrophic growth responses.