Fluorescence quenching and lifetime distributions of single molecules on glass surfaces

The fluorescence lifetimes of TMR molecules, attached covalently to glass surfaces while solvated in ethylene glycol, were measured in the absence and presence of energy transfer acceptors in solution. Fluorescence quenching by either the glass surface or acceptor molecules generates nonexponential decays in bulk samples. By means of single-molecule fluorescence lifetime microscopy, the corresponding lifetime distributions are obtained and found to be quasi-continuous. The moments of the survival times associated with the best stretched exponential fits to the bulk data are also compared with those calculated from the lifetime distributions and the agreement is not perfect with the stretched exponential yielding a distribution that is too asymmetric.