Photobleaching as a tool to measure the local strain field in fibrous membranes of connective tissues

Connective tissues are complex structures which contain collagen and elastin fibers. These fiber-based structures have a great influence on material mechanical properties and need to be studied at the microscopic scale. Several microscopy techniques have been developed in order to image such microstructures; among them are two-photon excited fluorescence microscopy and second harmonic generation. These observations have been coupled with mechanical characterization to link microstructural kinematics to macroscopic material parameter evolution. In this study, we present a new approach to measure local strain in soft biological tissues using a side-effect of fluorescence microscopy: photobleaching. Controlling the loss of fluorescence induced by photobleaching, we create a pattern on our sample that we can monitor during mechanical loading. The image analysis allows three-dimensional displacements of the patterns at various loading levels to be computed. Then, local strain distribution is derived using the finite element discretization on a four-node element mesh created from our photobleached pattern. Photobleaching tests on a human liver capsule have revealed that this technique is non-destructive and does not have any impact on mechanical properties. This method is likely to have other applications in biological material studies, considering that all collagen–elastin fiber-based biological tissues possess autofluorescence properties and thus can be photobleached.