CFM: confinement force microscopy-a dynamic, precise and stable microconfiner for traction force microscopy in spatial confinement.

Cells migrating through tissues experience changing physical confinement, yet methods to dynamically control confinement while quantifying the resulting forces remain limited. Here we present a microconfiner platform for live-cell imaging that enables programmable confinement, allowing real-time control over the level, timing and frequency of confinement while measuring traction forces exerted on the microenvironment, a method we term confinement force microscopy (CFM). Using CFM, we find that cells respond to confinement in two phases: a rapid passive stress rise caused by compression of the cell body and nucleus against the substrate, followed by an active stress increase associated with enhanced contractility, intracellular pressure buildup and bleb formation. Bleb expansion can partially relieve pressure and reduce stress on the surroundings. ROCK and myosin II inhibition both reduce stress generation, but with distinct effects on blebbing. Overall, CFM provides a versatile approach to study dynamic mechanical adaptation in tissue-like environments.