Biofilms are complex aggregations of microorganisms in which cells adhere on a surface and are held together in a matrix of extracellular polysaccharide substances (EPS). Biofilms are very resistant to attacks and are responsible for the majority of bacterial infections. They are highly heterogeneous and within colonies, both chemical and physical gradients exist. Therefore, in order to study and understand 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 presented and employed for the study of Pseudomonas aeruginosa biofilms. Using TRAST, we found anoxic zones within the colonies. We also observed that oxygen consumption extends outside the areas of high cell densities, establishing a gradient between the biofilm interior and air saturation at the exterior. 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. The tools presented here can offer information in high spatial resolutions that can be applied to address emerging questions about the biofilm mode of life.