Thanks to the development of new 3D Imaging techniques, volumetric data of thick samples, especially tissues, are commonly available. Several algorithms were proposed to analyze cells or nuclei in tissues, however these tools are limited to two dimensions. Within any given tissue, cells are not likely to be organized randomly and as such have specific patterns of cell-cell interaction forming complex communication networks. In this paper, we propose a new set of tools as an approach to segment and analyze tissues in 3D with single cell resolution. This new tool box can identify and compute the geographical location of single cells and analyze the potential physical interactions between different cell types and in 3D. As a proof-of-principle, we applied our methodology to investigation of the cyto-architecture of the islets of Langerhans in mice and monkeys. The results obtained here are a significant improvement in current methodologies and provides new insight into the organization of alpha cells and their cellular interactions within the islet's cellular framework.