Macrophage volume analysis and real-time monitoring by digital holography

SignificanceCell volume serves as a macroscopic indicator of cellular metabolism, growth, and signal transduction. Understanding how cell volume varies across macrophage phenotypes is essential for interpreting their functional states and activation processes.AimWe present a label-free, noninvasive method for 3D morphological characterization and volume quantification of macrophages and apply it to monitor volume variations during their biological behaviors.ApproachWe employed a home-made off-axis digital holographic microscopy (DHM) system based on a Mach–Zehnder interferometric configuration to reconstruct the 3D morphology of cells. Using the mathematical relationship between the phase map and the cell thickness, we further calculated their cellular volume.ResultsOur measurements show that M0 macrophages have a nearly spherical morphology with a volume of 970.54±153.15 μm3. M1 macrophages exhibit a flattened, pancake-like morphology with pseudopodia, with a significantly larger volume of 3822.00±437.45 μm3. M2 macrophages present a morphology resembling that of M0 cells but with a volume of 2743.10±254.67 μm3. It indicates that the cell volume might be a potential parameter to distinguish different polarized macrophages. In addition, we successfully monitored the volume changes of macrophages during cell death, cell division, and the polarization process from M0 to M1. The cell-death process behaved as expected, with the cell volume continuously decreasing and eventually reaching a plateau. The division event we observed occurred together with cell death; during division, the total volume of the cell cluster increased sharply and then gradually decreased as cell death progressed. During the 8-h M0-to-M1 polarization process, two phases of gradual volume increase and two phases of rapid volume increase were observed. In the early stage (0 to 2 h), M0 macrophages were likely not fully activated, showing only mild volume changes but still exhibiting an increasing trend. At 6 to 8 h, the cells undergo a sharp increase in volume, reflecting their full activation and transition toward the M1 phenotype.ConclusionsWe demonstrate that DHM offers a powerful label-free strategy for quantifying macrophage volume and capturing the volume dynamic. It might be a potential approach for a macroscopic characterization to observe intracellular activities.