Measurement of oxygen concentrations in bacterial biofilms using transient state monitoring by single plane illumination microscopy

Oxygen is required for respiration in aerobic bacteria and hence plays an essential role in the generation of energy necessary for cell maintenance and growth. The distribution of oxygen within a biofilm can be heterogeneous and may result in bacterial cells exhibiting distinct physiological states depending on their local oxygen availability. Here, we have employed transient state (TRAST) monitoring, a non-invasive method that can measure the relative populations of triplet states by timemodulated illumination, to determine the distribution of oxygen concentrations in biofilm colonies. The method was implemented on a single plane illumination microscope that can perform optical slicing, making it a suitable approach for imaging thick samples.Wepresent a map of triplet relaxation times measured inside a Pseudomonas aeruginosa biofilm at micrometre resolution.Wealso provide a calibration method that can evaluate oxygen concentration based on TRAST contrast. Our results indicate that anoxic zones exist within the colonies, and oxygen consumption extends outside the areas of high cell densities, establishing a gradient until levels return to air saturation.