Three Dimensional Dynamics of Epithelial Monolayers

Collective migration and pulsatile flows in epithelial monolayers are commonly quantified using projected area, implicitly assuming constant cell volume and prism-like cell geometry. These "21/2D" assumptions neglect the intrinsic three-dimensional height and volume dynamics that accompany density fluctuations in confluent, space-filling tissues. Here, we combine 2D quantitative phase imaging (QPI) and 3D refractive index tomography to obtain time-lapse maps of height, volume, and dry mass in Madin-Darby canine kidney (MDCK) epithelial monolayers undergoing collective motion. This is, to our knowledge, the first systematic use of QPI to quantify epithelial monolayer height, volume, and mass dynamics in situ. From independent measurements of refractive index and height, we determine an average dry mass concentration cd = 0.287 g/ml with 2% variability between cells and over time, demonstrating tight regulation of dry-mass density even during large-amplitude pulsations and density changes. The mean height of the monolayer increases with cell density, while the mean cell volume decreases, revealing contact inhibition of cell size. Pixel- and disc-wise statistics show broad, gamma-like height distributions and strong spatio-temporal height fluctuations that remain substantial at high cell density. Cell-resolved tracking demonstrates that height, area, and volume fluctuate synchronously, with volume changes dominated by area rather than height variations, while dry-mass density remains nearly constant. Dynamic structure-factor analysis reveals subdiffusive dynamics and propagating compression-decompression waves, and a continuum mass-flux analysis shows that the depth-averaged continuity equation fails on cellular scales and is restored only after spatial and temporal coarse-graining. Using simple geometrical models, we show that prismatoid cell shapes with constant true volume can reproduce the observed correlations between height, apical area, and "projected" volume, implying that non-prismatic cell geometry biases 21/2D estimates. Together, these results overturn the assumptions of mass/volume conservation and plug-flow-like monolayer kinematics at cellular scales, and highlight the need to incorporate dry-mass regulation and 3D cell shape into models of epithelial dynamics. SIGNIFICANCE STATEMENTUsing QPI, we provide the first comprehensive and time-resolved characterisation of epithelial monolayer height, volume, and dry mass in situ, yielding quantitative measures that both extend and revise earlier work based on 2D imaging alone. Our measurements challenge two long-standing assumptions in epithelial physics: that cell mass or volume is conserved on the timescales of collective motion, and that monolayers behave as "21/2D" plug-flow sheets with vertical, prism-like cells of equal apical and basal area. These findings necessitate a re-examination of prior experimental interpretations and a reassessment of when existing continuum and cell-based models faithfully describe epithelial monolayer dynamics. They also provide benchmarks for future 3D theories and experiments.