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

In vivo, cells experience complex tissue environments with various chemical and physical features. They sense and respond to tissue morphology and mechanical properties and adjust their behavior and function based on the surrounding. In contrast to the free environment experienced on 2D substrates commonly used in research, the 3D natural environment represents a major physical obstacle for cells. Here, cells are usually confined either by the extracellular matrix (ECM) or neighboring cells. The importance of such confinements has been demon-strated in the past decades by showing its influence on cell decision-making in many vital biological processes such as migration, division and cytoskeletal reorganization. Despite these insights, the sheer level of complexity faced when studying cell biological questions in biomimetic confined situations, led to an indispensable need for a 3D system which can simulate the in vivo confined condition, while being capable of providing microenvironments with different chemical and physical properties for the cells and capturing the mechanical forces and properties of the studied biological sample. Here we introduce a microconfiner that finally provides a new imaging capacity, namely the confine-ment force microscopy (CFM). We are able to adjust the confinement level in real time during microscopy while measuring not only the the cellular traction but also the cellular compression forces. Furthermore, the chemical and physical properties of the microenvironment can be optimized for the respective questions. We demonstrate the power of this confinement system by the mechanical response of cells, migration analysis of immune cells, the timed force generation during durotaxis driven adhesion switching and the viscoelastic properties of cancer tissue.