Continuous, high-speed 3D topographic profiling of living tissue monolayers

<jats:p>Tissue monolayers are active living matter whose three-dimensional (3D) architecture and mechanical remodeling regulate tissue function, yet fast and continuous topographic monitoring remains difficult. Here, we present FLuorescence EXclusion microscopy fOr Monolayers (FLEXOM), a microfluidic platform that combines multilayer micropillar arrays with negative-staining optics to convert wide-field images into self-calibrated 3D height maps. Geometry-anchored reference cavities maintain in-frame calibration standards even in fully confluent monolayer fields, enabling continuous 3D profiling from isolated cells to confluent sheets with sub-second temporal resolution (~250 ms), sub-micrometer axial precision (~0.37 μm), and multi-day biocompatibility and operational stability (&gt;96 h). FLEXOM reveals two distinct anisotropic signatures in tissue monolayers. First, monolayers of different cell types show distinct vertical thickness profiles despite comparable lateral footprints. Only two vertical descriptors, mean height and height variability, distinguished cell types more accurately than nine 2D lateral descriptors, including area, circularity, and convexity (95% vs. 76%). Second, under acute osmotic shock, confluent monolayers exhibit geometrically anisotropic and temporally decoupled vertical-lateral remodeling: height changes dominate the overall volume response and precede lateral remodeling. Surprisingly, cyclic isotonic-hypotonic pulses every 3 min confine remodeling entirely to the vertical axis. Overall, our work provides a high-resolution 3D profiling tool and a framework for axis-resolved analysis of topographic responses in active living matter.</jats:p>