Self-referenced RGB colour imaging of intracellular oxygen

Intracellular oxygen is an important indicator for cell metabolism and respiration. We have designed self-referenced RGB PEBBLEs that enable a simple readout of the intracellular oxygen distribution with conventional wide-field microscopy and a standard RGB digital camera. The RGB PEBBLEs consist of a hydrophobic matrix covered with amino groups on the surface to confer water-dispersibility. Two luminophores are incorporated in a hydrophobic polystyrene matrix that is highly permeable to oxygen. In polystyrene, the dyes are largely protected from quenching or aggregation by cellular components. The dyes have been selected to match the green and the red channel of digital cameras. While the red emission of the oxygen probe is highly sensitive to oxygen with a quenching response of 74%, the green emission of the reference dye is stable under varying oxygen concentrations. Ratiometric images of intracellular oxygen have been acquired that are inherently resistant to fluctuations in absolute signal intensities. As RGB PEBBLEs respond within seconds to changing oxygen concentrations, they are amenable to monitoring fast cellular dynamics.