Direct observation of the triplet lifetime quenching of single dye molecules by molecular oxygen

The influence of oxygen on the photophysical properties of individual DiI18 molecules has been investigated by means of both wide field and confocal scanning optical fluorescence microscopy. Excited close to saturation intensity, single-molecule fluorescence detected in wide field by a charge-coupled device camera showed a dramatic increase at exposure to air compared to the fluorescence when the sample was protected from oxygen by nitrogen flush. The change of the triplet lifetime of individual dye molecules due to oxygen quenching was measured in real time by means of time resolved single photon counting. In the presence of oxygen, the triplet state lifetime decreases from several tens of milliseconds down to fractions of a millisecond, whereas no changes of the intersystem crossing quantum yield and the fluorescence lifetime are observed. The triplet lifetimes in the presence and absence of oxygen, respectively, are anti-correlated indicative of heterogeneity of the polymer surrounding the dye molecules.