Fluorescence Lifetime Imaging Detects Long-Lifetime Signal Associated with Reduced Pyocyanin at the Surface of <i>Pseudomonas aeruginosa</i> Biofilms and in Cross-Feeding Conditions

Understanding bacterial physiology in real-world environments requires noninvasive approaches and is a challenging yet necessary endeavor to effectively treat infectious disease. Bacteria evolve strategies to tolerate chemical gradients associated with infections. The DIVER (Deep Imaging Via Enhanced Recovery) microscope can image autofluorescence and fluorescence lifetime throughout samples with high optical scattering, enabling the study of naturally formed chemical gradients throughout intact biofilms. Using the DIVER, a long fluorescent lifetime signal associated with reduced pyocyanin, a molecule for electron cycling in low oxygen, was detected in low-oxygen conditions at the surface of <i>Pseudomonas aeruginosa</i> biofilms and in the presence of fermentation metabolites from <i>Rothia mucilaginosa</i>, which cocolonizes infected airways with <i>P. aeruginosa</i>. These findings underscore the utility of the DIVER microscope and fluorescent lifetime for noninvasive studies of bacterial physiology within complex environments, which could inform on more effective strategies for managing chronic infection.