Multicellular aggregates contain chemical gradients that promote physiological differentiation, and this contributes to drug tolerance in structures such as tumors and infectious biofilms. We study the strategies that enable survival of bacteria in multicellular structures. For the pathogen Pseudomonas aeruginosa, these include the use of specialized metabolic pathways and the control of multicellular organization in ways that promote access to resources. My group uses diverse techniques to study mechanisms that determine the physiology and topography of P. aeruginosa aggregates. Microelectrode profiling and fluorescent reporters show that chemical subzones within P. aeruginosa biofilms correlate with localized metabolic gene expression. Using Stimulated Raman scattering microscopy we can profile metabolic activity along chemical gradients, and we have uncovered a complex interplay of metabolic strategies. These include the cross-feeding of metabolites, such as endogenous lactate and phenazines. We have also shown that, in addition to promoting redox homeostasis, phenazine metabolism in particular has the effect of enhancing antibiotic tolerance for cells in biofilms. Using fluorescent labeling and high-resolution microscopic analysis, we have also been able to visualize depth-dependent effects on cellular arrangement and virulence factor production in biofilms. We have found that cells in biofilms form distinct subzones of spatial organization that affect the distribution of resources across the structure. Furthermore, localization within the biofilm also affects the production of a newly discovered virulence factor called the R-body, a large extendable polymer that is toxic to eukaryotic hosts. Together, these observations reveal the diverse ways in which multicellular physiology and behavior can enhance the robustness and pathogenicity of biofilm-based P. aeruginosa infections. Techniques that reveal the chemical and spatial variation within multicellular structures across depth provide insights that are specific to the communal lifestyle, and these insights have the potential to inform efforts to treat recalcitrant diseases.