Determining the mechanical properties of biological samples using Atomic Force Microscopy (AFM) has opened new possibilities for the characterization of biological materials and relative applications [1].The AFM indentation is the most extensively used method for the nanomechanical characterization of biological samples at the nanoscale; it can be equally applied from the characterization of single molecules and proteins to the characterization of complex biological samples such as cells and tissues [1]. However, the biological materials’ characterization using AFM is still considered as a challenging procedure; many errors may arise mostly related to data processing and the misuse of contact mechanics models that consider the sample as an elastic half space (biological samples at the nanoscale are highly heterogeneous and non-isotropic materials) [1-2].Nevertheless, a new contact mechanics theory was recently developed; the average Young’s modulus theory by members of the research team[2]. The aforementioned theory may be used for a complete mechanical characterization of any single region at the nanoscale; thus, it can reveal the depth-dependent mechanical properties of biological samples.The basic goal of this project is to use the average Young’s modulus theory for a 3-dimensional (3D) mechanical characterization of biological samples at the nanoscale.It is significant to note that the mechanical properties of biological samples vary significantly as the indentation depth increases[2]; as a result,currently existing methods do not take this into account and their results depend on the respective AFM user selecting the maximum indentation depth.In other words, existing AFM methods provide a 2D characterization (since they do not take into account the mechanical properties’ alterations in the 3rddimension, i.e., as the indentation depth increases and their results are user dependent.On the contrary, the proposed project aims towards the development of a software package (based on the average Young’s modulus theory)able to derive3D mechanical patterns of any biological sample. The 3D characterization will be also applied on normal and cancer cells as a test of the software performance.The aforementioned samples are of out most scientific interest related to early cancer diagnosis[3] and other applications [4].A software package based on the average Young’s modulus theory will be developed in order to correctly process the force curves(force–indentation AFM data)of highly heterogeneous materials(e.g. cells). The software will be able to derive the average Young’s modulus at different nano regions and for different indentation depths. As a result using multiple average Young’s modulus values at different location on the xyz dimensional space will lead to a 3D mechanical characterization. To test the validity of the software experiments on reference samples (agarose gels) will be performed first. Subsequently, normal and cancerous cells from mice will be tested in order to derive accurate mechanical 3D mechanical patterns in each case. Thus, the basic limitation of AFM techniques which is the ability to acquire only 2-dimensionalinformationregarding the sample’s mechanical properties will be overcome. Special attention will be also given to avoid increasing the complexity of the standard AFM experimental procedures.