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Most microbes can produce surface-associated or suspended aggregates called biofilms, which are encased within a biopolymer-rich matrix. The biofilm matrix provides structural integrity to the aggregates and shields the resident cells against environmental stressors, including antibiotic treatment. Microscopy permits examination of biofilm structure in relation to the spatial localization of important biofilm matrix components. This review highlights microscopic approaches to investigate bacterial biofilm assembly, matrix composition, and localization using  Pseudomonas aeruginosa  as a model organism. Initial microscopic investigations provided information about the role key matrix components play in elaborating biofilm aggregate structures. Additionally, staining of matrix components using specific labels revealed distinct positioning of matrix components within the aggregates relative to the resident cells. In some cases, it was found that individual matrix components co-localize within aggregates. The methodologies for studying the biofilm matrix are continuing to develop as our studies reveal novel aspects of its composition and function. We additionally describe some outstanding questions and how microscopy might be used to identify the functional aspects of biofilm matrix components.

Confocal Laser Scanning Microscopy for Analysis of Pseudomonas aeruginosa Biofilm Architecture and Matrix Localization