Advanced 3D Image Analysis of Bacterial Biofilm Architecture Tuberculosis (TB) remains a leading cause of death from infectious diseases globally. A critical factor in the pathogenesis and drug tolerance of Mycobacterium tuberculosis (Mtb) is its ability to organize into biofilms, which are complex three-dimensional structures characterized by high cellular density and the formation of "cords". Confocal Laser Scanning Microscopy (CLSM) is the preferred tool for studying these architectures; however, automated quantitative analysis is severely limited by imaging artifacts. This thesis focuses on the analysis of a dataset characterized by severe anisotropy and marked Z-axis undersampling. Under these conditions, the elongation of the Point Spread Function (PSF) generates a distorted representation of the bacteria, preventing accurate manual annotations (Ground Truth).