Can Single Molecule Fluorescence Measurements Be Biased? Studies of Porphyrin Isomers Embedded in Non-Degassed Polymer Matrices

Detection of single molecules via fluorescence is a technique widely used in chemistry, physics, and biology. It has been generally recognized that the observation of single molecules by fluorescence is limited to fluorophores with large emission and small triplet formation quantum yields. However, single molecule fluorescence has been reported also for molecules known for efficient triplet state population. We solve this puzzle by demonstrating that such unusual behavior can be explained by the presence of oxygen in the vicinity of a single molecule emitter. Single molecule fluorescence lifetimes of a porphyrin derivative and its isomers, porphycene and hemiporphycene in poly(methyl methacrylate) films are several times shorter than the lifetimes measured for bulk samples in the same polymer matrix. Similar quenching of fluorescence lifetimes by oxygen is observed for porphycene embedded in cryogenic nitrogen and argon matrices containing increasing amounts of oxygen. Simulations of the emission rate as a function of triplet lifetime show that single molecules characterized by considerable triplet formation yields can only be detected via fluorescence if their triplet lifetime is shortened below a certain limit by the presence of a quencher, such as oxygen. Thus, only a fraction of molecules present in the sample is observed, those with sufficiently short triplet lifetimes. We postulate that such bias can often be encountered under conditions of rigid matrices or degassed solutions.