Experimental and computational dynamic force spectroscopy is widely used to determine the mechanical properties of single biomolecules. Whereas so far the focus has mainly been on rupture or unfolding forces, recent force-probe molecular dynamics simulations have revealed a strong loading rate dependence of biomolecular elasticities, which cannot be explained by the established one-dimensional transition-state treatments. We show that this nonequilibrium behavior can be explained by a theory that includes relaxation effects. For three structurally and mechanically quite diverse systems, a single relaxation mode suffices to quantitatively describe their loading-rate-dependent elastic behavior. Atomistic simulations of these systems revealed the microscopic nature of the respective relaxation modes. This result suggests a new type of ``elasticity spectroscopy'' experiment, which should render nonequilibrium properties of structured macromolecules accessible to single-molecule force spectroscopy.

Development of neuronal tissue, such as folding of the brain, and formation of the fovea centralis in the human retina are intimately connected with the mechanical properties of the underlying cells and the extracellular matrix. In particular for neuronal tissue as complex as the vertebrate retina, mechanical properties are still a matter of debate due to their relation to numerous diseases as well as surgery, where the tension of the retina can result in tissue detachment during cutting. However, measuring the elasticity of adult retina wholemounts is difficult and until now only the mechanical properties at the surface have been characterized with micrometer resolution. Many processes, however, such as pathological changes prone to cause tissue rupture and detachment, respectively, are reflected in variations of retina elasticity at smaller length scales at the protein level. In the present work we demonstrate that freely oscillating cantilevers composed of nanostructured TiO₂ scaffolds can be employed to study the frequency-dependent mechanical response of adult mammalian retina explants at the nanoscale. Constituting highly versatile scaffolds with strong tissue attachment for long-term organotypic culture atop, these scaffolds perform damped vibrations as fingerprints of the mechanical tissue properties that are derived using finite element calculations. Since the tissue adheres to the nanostructures via constitutive proteins on the photoreceptor side of the retina, the latter are stretched and compressed during vibration of the underlying scaffold. Probing mechanical response of individual proteins within the tissue, the proposed mechanical spectroscopy approach opens the way for studying tissue mechanics, diseases and the effect of drugs at the protein level.