Design and Application of an Optical Sensor for Simultaneous Imaging of pH and Dissolved O2 with Low Cross-Talk

Visualization and quantification of analytes such as O-2 or pH is essential in biological research. Here we present the design and application of a new optical dual-analyte sensor for imaging, optimized to have low cross-sensitivity between the two analytes O-2 and pH. The used indicator and reference dyes were selected to match the different channels of a commercial 2CCD (RGB + NIR) camera. A red-light emitting O-2-sensitive europium complex (Eu(HPhN)(3)dpp) with a dynamic range of 0-20% O-2 in the finished sensor was combined with a near-infrared emitting pH indicator (OHButoxy-aza-BODIPY) with a dynamic range of pH 7.2-8.8. To enable ratiometric readout, an inert reference coumarin dye (Bu(3)Coum) was co-immobilized with the optical indicators. In order to maximize the sensor signal, inert diamond powder was added to one sensor layer as a simple way to increase scattering of light within the sensor. Furthermore, the addition of an optical isolation layer enabled measurements in highly fluorescent samples, such as algal biofilms. The sensor was tested in a marine photosynthetic microbial mat.