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

The influence of oxygen on the photophysical properties of individual DiI(1 8) molecules has been investigated by means of both wide field and confocal scanning optical fluorescence microscopy. Excited close to saturation inte nsity, single-molecule fluorescence detected in wide field by a charge-coup led device camera showed a dramatic increase at exposure to air compared to the fluorescence when the sample was protected from oxygen by nitrogen flu sh. The change of the triplet lifetime of individual dye molecules due to o xygen quenching was measured in real time by means of time resolved single photon counting. In the presence of oxygen, the triplet state lifetime decr eases from several tens of milliseconds down to fractions of a millisecond, whereas no changes of the intersystem crossing quantum yield and the fluor escence lifetime are observed. The triplet lifetimes in the presence and ab sence of oxygen, respectively, are anti-correlated indicative of heterogene ity of the polymer surrounding the dye molecules. (C) 2001 American Institu te of Physics.