AFM can be successfully applied for comprehensive nano-mechanical characterization of biomaterials, cells and tissues, under near physiological conditions. Currently, the trend is to extend this by studying the mechanobiology of living cells while evaluating their structure and the interaction with their cell culture substrates. In particular, it is interesting to understand how cell behaviour is driven by the cytoskeletal dynamics and cell mechanics in typical cell culture scaffold scenarios. We will show automated large area multiparametric characterization of densely packed cell layers and highly corrugated tissue samples. We will discuss how these, in combination with advanced optical microscopy techniques, can overcome the inherent drawbacks of traditional AFM systems for characterizing challenging biological samples.

Cells adapt their shape and react to the surrounding environment by a dynamic reorganization of the F-actin cytoskeleton. We will demonstrate how cell spreading and migration in living KPG-7 fibroblasts and CHO cells, can be studied with high-speed AFM and associated with spatially resolved cytoskeletal reorganization events. We will further extend this with high-speed mechanical mapping of confluent cell layers, which in combination with optical tiling can be applied to automated analysis of large sample areas.

We also studied the assembly kinetics and structural hierarchy of collagen I, as one of the most used proteins in tissue engineering. We applied high-speed AFM imaging with a temporal resolution on the second to millisecond scale to resolve dynamic processes such as the collagen fibrillogenesis. We will provide insight into the structural formation of collagen type I, emphasizing the intermediate steps in the process.

In the past, investigating large and rough samples such as tissues and hydrogels using AFM was challenging due to the limited z-axis of the AFM. Using osteoarthritic cartilage as an example, we will demonstrate multi-region AFM probing over a large, rough sample area while providing additional correlative optical data sets.

External mechanical stress is known to influence cell mechanics in correlation to the differences in actin cytoskeleton dynamics. As a tool for analysing the complex cellular mechanobiology, we went beyond purely elastic models, and performed sine oscillations (up to 500Hz, amplitude 5-60 nm) in Z while in contact with the surface to probe the frequency-dependent response of living fibroblasts. We will further discuss how to calculate the viscoelastic properties, characterized by the dynamic storage and loss modulus (E’, E’’) distribution in such samples.