Rate-independent hysteretic energy dissipation in collagen fibrils.

Nanoindentation cycles measured with an atomic force microscope on hydrated collagen fibrils exhibit a rate-independent hysteresis with return point memory. This previously unknown energy dissipation mechanism describes in unified form elastoplastic indentation, capillary adhesion, and surface leveling at indentation velocities smaller than 1 µm s-1, where viscous friction is negligible. A generic hysteresis model, based on force-distance data measured during one large approach-retract cycle, predicts the force (output) and the dissipated energy for arbitrary indentation trajectories (input). While both quantities are rate independent, they do depend nonlinearly on indentation history and on indentation amplitude.

Probing local lateral forces of focal adhesions and cell-cell junctions of living cells by torsional force spectroscopy.

The number and strength of mechanical connections of cells to their local environment can be indicative of their migration and invasion potential. Gaining direct access to the mechanical properties of individual connections and bringing them into a relationship with the state of disease, however, is a formidable task. Here, we present a method to directly sense focal adhesions and cell-cell contacts with a force sensor to quantify the lateral forces of their anchoring points. We found local lateral forces of 1.0-1.5 nN for focal adhesions and slightly higher values at the interfaces between cells where cell-cell contacts are located. Interestingly, a modified surface layer was observed exhibiting considerably reduced tip friction directly next to the area of a retracting cell edge on the substrate. We expect that this technique can improve the understanding of the relationship between mechanical properties of cell connections and the pathological state of cells in the future.

Nanomechanical 3D Depth Profiling of Collagen Fibrils in Native Tendon.

Connective tissue displays a large compositional and structural complexity that involves multiple length scales. In particular, on the molecular and the nanometer level, the elementary processes that determine the biomechanics of collagen fibrils in connective tissues are still poorly understood. Here, we use atomic force microscopy (AFM) to determine the three-dimensional (3D) depth profiles of the local nanomechanical properties of collagen fibrils and their embedding interfibrillar matrix in native (unfixed), hydrated Achilles tendon of sheep and chickens. AFM imaging in air with controlled humidity preserves the tissue's water content, allowing the assembly of collagen fibrils to be imaged in high resolution beneath an approximately 5-10 nm thick layer of the fluid components of the interfibrillar matrix. We collect pointwise force-distance (FD) data and amplitude-phase-distance (APD) data, from which we construct 3D depth profiles of the local tip-sample interaction forces. The 3D images reveal the nanomechanical morphology of unfixed, hydrated collagen fibrils in native tendon with a 0.1 nm depth resolution and a 10 nm lateral resolution. We observe a diversity in the nanomechanical properties among individual collagen fibrils in their adhesive and in their repulsive, viscoelastic mechanical response as well as among the contact points between adjacent collagen fibrils. This sheds new light on the role of interfibrillar bonds and the mechanical properties of the interfibrillar matrix in the biomechanics of tendon.

Nanomechanical sub-surface mapping of living biological cells by force microscopy.

Atomic force microscopy allows for the nanomechanical surface characterization of a multitude of types of materials with highest spatial precision in various relevant environments. In recent years, researchers have refined this methodology to analyze living biological materials in vitro. The atomic force microscope thus has become an essential instrument for the (in many cases) non-destructive, high-resolution imaging of cells and visualization of their dynamic mechanical processes. Mapping force versus distance curves and the local evaluation of soft samples allow the operator to "see" beneath the sample surface and to capture the local mechanical properties. In this work, we combine atomic force microscopy with fluorescence microscopy to investigate cancerous epithelial breast cells in culture medium. With unprecedented spatial resolution, we provide tomographic images for the local elasticity of confluent layers of cells. For these particular samples, a layer of higher elastic modulus located directly beneath the cell membrane in comparison with the average elastic properties was observed. Strikingly, this layer appears to be perforated at unique locations of the sample surface of weakest mechanical properties where distinct features were visible permitting the tip to indent farthest into the cell's volume. We interpret this layer as the cell membrane mechanically supported by the components of the cytoskeleton that is populated with sites of integral membrane proteins. These proteins act as breaking points for the indenter thus explaining the mechanical weakness at these locations. In contrast, the highest mechanical strength of the cell was found at locations of the cell cores as cross-checked by fluorescence microscopy images of staining experiments, in particular at nucleoli sites as the cumulative elastic modulus there comprises cytoskeletal features and the tight packing ribosomal DNA of the cell.

3D depth profiling of the interaction between an AFM tip and fluid polymer solutions.

In the atomic force microscopy (AFM) investigation of soft polymers and liquids, the tip-sample interaction is dominated by long-range van der Waals forces, capillary forces and adhesion. Furthermore, the tip can indent several tens of nanometres into the surface, and it can pull off a polymer filament from the surface. Therefore, measuring the unperturbed shape of a polymeric fluid can be challenging. Here, we study the tip-sample interaction with polystyrene droplets swollen in chloroform vapour, where we can utilize the solvent vapour concentration to adjust the specimen's mechanical properties from a stiff solid to a fluid film. With the same AFM tip, we use two different AFM force spectroscopy methods to measure three-dimensional (3D) depth profiles of the tip-sample interaction: force-distance (FD) curves and amplitude-phase-distance (APD) curves. The 3D depth profiles reconstructed from FD and APD measurements provide detailed insight into the tip-sample interaction mechanism for a fluid polymer solution. The fluid's intrinsic relaxation time, which we measure with an AFM-based step-strain experiment, is essential for understanding the tip-sample interaction mechanism. Furthermore, measuring 3D depth profiles and using APD data to reconstruct the unperturbed surface comprise a versatile methodology for obtaining accurate dimensional measurements of fluid and gel-like objects on the nanometre scale.