NOVEL SPECTROSCOPY TECHNIQUES FOR FUNCTIONAL IMAGING OF BACTERIAL BIOFILMS AT MICROSCOPIC LEVEL

Abstract

Biofilms are complex aggregations of microorganisms that are responsible for the majority of bacterial infections. Within colonies, both chemical and physical gradients exist. Therefore, in order to study their functionalities, it is essential to develop methods that can provide information at high spatiotemporal resolutions. In this thesis, two novel spectroscopic techniques, namely Transient State (TRAST) monitoring and Brillouin Imaging – that can optically measure oxygen distributions and stiffness of the sample, respectively – are used for the study of Pseudomonas aeruginosa biofilms. We found anoxic zones within the colonies and oxygen gradients inside and outside the colonies. Using Brillouin microscopy, we found two distinct stiffness patterns in colonies: in younger colonies, stiffness increased towards their interior, whereas, some larger (> 45 μm in diameter) colonies were found to have less stiff interiors, suggesting they were hollow, or possibly undergoing transition to the seeding dispersal stage.