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Bacterial biofilms are groups of bacteria that exist within a self-produced extracellular matrix, adhering to each other and usually to a surface. They grow on medical equipment and inserts such as catheters, and are responsible for many persistent infections throughout the body, as they can have high resistance to many antimicrobials. Pseudomonas aeruginosa is an opportunistic pathogen that can lead to both acute and chronic infections, and is used as a model for research into biofilms. Direct biochemical methods of imaging molecules in bacterial biofilms are of high value in gaining a better understanding of the fundamental biology of biofilms, and biochemical gradients within them. TOF-SIMS is one approach, which combines relatively high spatial resolution and sensitivity, and can perform depth profiling analysis. It has been used to analyze bacterial biofilms, but has not yet been applied to study the distribution of antimicrobials (including antibiotics and the antimicrobial metal gallium) within biofilms. Here we have compared two methods of imaging the interior structure of P. aeruginosa in biological samples using TOF-SIMS, looking at both antimicrobials and endogenous biochemicals: cryosectioning of tissue samples, and depth profiling to give pseudo-3D images. The sample types included both simple biofilms grown on glass slides, and bacteria growing in tissues in an ex-vivo pig lung model. The two techniques for the 3D imaging of biofilms are potentially valuable, complementary tools for analyzing bacterial infection.