Bacterial biofilms consist of surface adherent bacteria that surround themselves with a polymer matrix that provides environmental protection and antibiotic resistance. Biofilms can grow on most implanted medical devices, on heart valves, and in the lungs of patients with cystic fibrosis, resulting in difficult to treat infections that an become blood-borne and spread throughout the body. Understanding the physical properties of biofilms is therefore of interest as such insight may lead to methods for their disruption and removal. Biofilms have been characterized biochemically, as the general composition of the matrix is known, as are the specific polysaccharides forming the bulk of the matrix for some species. Insight into physical properties of biofilms, such as elasticity and deformability, has been limited to macroscale techniques that assess averaged values. These techniques do not provide details on the spatial gradients of physical properties within a biofilm. Particle trackin is a technique in which multiple microbeads are placed in a material and observed using microscopy, allowing for the determination of physical properties and structure of the material they are embedded in. In this project, particle tracking will be used to assess various living biofilm systems. The overall goal is to gain an understanding of the structure, physical properties, and dynamics of biofilms, and to understand the effects of modulations in age and genetics of the organisms or environmental conditions. The first specific aim is to develop a system for probing the structure and properties of biofilms by using particle tracking to understand a model biofilm system. This will be accomplished by adding microbeads of various sizes and charges to a biofilm either while it is growing or after it has been formed, and then using the motion of the beads to characterize the system. The second specific aim is to actively move beads through a biofilm to understand biofilm dynamics and response to internal perturbation. Magnetic beads will be embedded into a biofilm and moved via magnetic tweezers to deform biofilm and measure how quickly it loses memory of the deformation. In addition, by using fluorescent bacteria it will be possible to analyze if applied internal forces result in bacterial displacement and motion, indicating that it may be possible to activate them within a biofilm. The third and fourth specific aims involve using particle tracking to understand internal perturbation of biofilm structure via the analysis of bacterial strains lacking matrix components and external perturbation of biofilm structure through the addition of known dispersal agents. The technique developed in the first aim will be used to assess the structure of these biofilms as compared to native biofilms in order to understand how to best compromise the integrity of biofilm structure.