Mechanical properties of soft biological tissues are key to the functionality of such tissues. Indentation has become a leading technique for characterizing the localized mechanical properties of materials, and it is an increasingly popular technique for studying biological materials. This thesis investigates difficulties and challenges regarding the application of micro-scale indentation.

This study first develops a novel approach, namely the multi-indent approach (MIA), which mitigates the long-standing challenge of surface detection and broadens the use of instrumented indentation for soft materials. The MIA is experimentally validated on isotropic polyacrylamide gel with load-unload cycles in indentation. Furthermore, it is applied to accurate characterization of poroelastic properties and allows for the use of much smaller probes and indentation depths for all measurements.

This study also establishes a corrected method for non-circular contact between a spherical tip and the transversely isotropic material. It develops experimental methods to determine the indentation moduli of a locally transversely isotropic collagenous material as well as the correlation between the aspect ratios (ARs) of contact and correction coefficients for contact areas (RCA).

Lastly, this study designs a method for selecting the spherical tip size for indentation based on the collagen fiber distribution in order to correctly sense the differences of local mechanical properties in heterogeneous tissues caused by the uneven fiber distribution. This thesis introduces methods and results that provide guidance and insight regarding the correct design and analysis of indentation experiments on soft materials and anisotropic biological tissues.