A simple optode based method for imaging O₂ distribution and dynamics in tap water biofilms

A ratiometric luminescence intensity imaging approach is presented, which enables spatial O₂ measurements in biofilm reactors with transparent planar O₂ optodes. Optodes consist of an O₂ sensitive luminescent dye immobilized in a 1-10 μm thick polymeric layer on a transparent carrier, e.g. a glass window. The method is based on sequential imaging of the O₂ dependent luminescence intensity, which are subsequently normalized with luminescent intensity images recorded under anoxic conditions. We present 2-dimensional O₂ distribution images at the base of a tap water biofilm measured with the new ratiometric method and compare the results with O₂ distribution images obtained in the same biofilm reactor with luminescence lifetime imaging. Using conventional digital cameras, such simple normalized luminescence intensity imaging can yield images of 2-dimensional O₂ distributions with a high signal-to-noise ratio and spatial resolution comparable or even surpassing those obtained with expensive and complex luminescence lifetime imaging systems. The method can be applied to biofilm growth incubators allowing intermittent experimental shifts to anoxic conditions or in systems, in which the O₂ concentration is depleted during incubation.