Multifocal optical projection microscopy enables label-free 3D measurement of cardiomyocyte cluster contractility.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) models have become an attractive tool for in vitro cardiac disease modeling and drug studies. These models are moving towards more complex three-dimensional microphysiological organ-on-chip systems. Label-free imaging-based techniques capable of quantifying contractility in 3D are needed, as traditional two-dimensional methods are ill-suited for 3D applications. Here, we developed multifocal (MF) optical projection microscopy (OPM) by integrating an electrically tunable lens to our in-house built optical projection tomography setup for extended depth of field brightfield imaging in CM clusters. We quantified cluster biomechanics by implementing our previously developed optical flow-based CM video analysis for MF-OPM. To demonstrate, we acquired and analyzed multiangle and multifocal projection videos of beating hiPSC-CM clusters in 3D hydrogel. We further quantified cluster contractility response to temperature and adrenaline and observed changes to beating rate and relaxation. Challenges emerge from light penetration and overlaying textures in larger clusters. However, our findings indicate that MF-OPM is suitable for contractility studies of 3D clusters. Thus, for the first time, MF-OPM is used in CM studies and hiPSC-CM 3D cluster contraction is quantified in multiple orientations and imaging planes.