Equibiaxial Stretching Device for High Magnification Live-Cell Confocal Fluorescence Microscopy

Cells are continuously exposed to mechanical forces in physiological and pathological situations, including a variety of tensile and compressive stresses. There is very active research exploring how cells and tissues respond and adapt to such stresses and how these responses integrate with mechanochemical signaling. This has generated a need for sophisticated tools compatible with standard cell culture protocols and microscopy methods, reproducing physiological stresses in in-vitro studies. This study presents the design, function, and characterization of a stretching device compatible with high-resolution optical and fluorescence microscopy. Numerous stretching devices, either pneumatic- or motor-based, have been developed and used in the field. We present one of these systems in detail, including design guidelines, a variety of applications, and all the tools to fabricate a similar setup. The system is based on a deformable polydimethylsiloxane (PDMS) membrane, stretched equibiaxially upon vacuum application, rendering a homogeneous, reproducible, and controlled sample strain. It provides a variety of tensile stresses, from punctual and immediate stretch to repeated stretch-release cycles of controlled amplitude and frequency. Substrate coating with adhesion proteins allows seeding cells bearing fluorescent reporters in the stretching device and performing live-imaging of these cells upon stretch using high magnification fluorescence microscopes. Compressive stresses can also be applied by letting the sample adapt to stretch and subsequently releasing it or by seeding the sample on a pre-stretched substrate before stretch release. Additional topographical patterning of the PDMS substrate enables imaging of the same sample in different microscopy modes (such as fluorescence and electron microscopy). Polyacrylamide gels can also be attached to the PDMS membrane, resulting in the stretching of cells seeded on substrates of different stiffnesses. Overall, by applying controlled tensile stresses on live samples, this stretching device, coupled with high-quality fluorescence microscopy, can address a large variety of questions in mechanobiology.