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Combined Imaging of Bacteria and Oxygen in Biofilms

ABSTRACT Transparent sensors for microscopic O2 imaging were developed by spin coating an ultrathin (<1- to 2-μm) layer of a luminescent O2 indicator onto coverslips. The sensors showed (i) an ideal Stern-Volmer quenching behavior of the luminescence lifetime towards O2 levels, (ii) homogeneous measuring characteristics over the sensor surface, and (iii) a linear decline of luminescence lifetime with increasing temperature. When a batch of such coverslip sensors has been characterized, their use is thus essentially calibration free at a known temperature. The sensors are easy to use in flow chambers and other growth devices used in microbiology. We present the first application for combined imaging of O2 and bacteria in a biofilm flow chamber mounted on a microscope equipped with a spinning-disk confocal unit and a luminescence lifetime camera system.