https://phys.org/news/2018-07-tools-characterise-physical-properties-biofilms.html

Oxygen plays an essential role in the generation of energy for cell maintenance and growth. Quantifying the amount of oxygen is necessary to study its effects during the various stages of biofilm growth. Current tools for measuring oxygen levels in biofilms either consume oxygen themselves (leading to less accurate results) or can only obtain accurate measurements from the surface but not within the biofilms.

To overcome these limitations, Prof Thorsten WOHLAND from the Departments of Biological Sciences and Chemistry, NUS together with Prof Yehuda COHEN and Prof Scott RICE from NTU have adapted a non-invasive technique called Transient State (TRAST) imaging and applied it to quantify oxygen levels in bacterial biofilms. This led to the identification of oxygen deficient zones within the microscopic colonies of P. aeruginosa. TRAST is a luminescence based imaging technique. It is based on the fact that certain fluorophores (a type of fluorescent chemical compound) occupy two different states, one which emits fluorescence and the other a non-fluorescent dark state. The fraction of fluorophores in the dark state depends on how often the fluorophores are excited and whether they are given enough time to come back from the dark states to the fluorescent states. By changing the illumination scheme in defined ways, the amount of fluorophores across the biofilm (in the dark state) can be measured easily. The measurements depend only on the fraction of fluorophores in the dark state, meaning that the accuracy is not affected even if certain regions in the biofilm have higher fluorophore concentration. Since oxygen suppresses the occupation of dark states, and thus lowers the fluorophores that reside in the dark state, TRAST can be used to quantify oxygen concentrations.