An experimental comparison of the maximum likelihood estimation and nonlinear least squares fluorescence lifetime analysis of single molecules

Two procedures based on the weighted least-squares (LS) and the maximum lik elihood estimation (MLE) method to confidently analyze single-molecule (SM! fluorescence decays with a total number (N) of 2500-60 000 counts have bee n elucidated and experimentally compared by analyzing measured bulk and SM decays. The key observation of this comparison is that the LS systematicall y underestimates the fluorescence lifetimes by similar to5%, for the range of 1000-20 000 events, whereas the MLE method gives stable results over the whole intensity range, even at counts N less than 1000, where the by analy sis delivers unreasonable values. This difference can be attributed to the different statistics approaches and results from improper weighting of the LS method. As expected from theory, the results of both methods became equi valent above a certain threshold of N detected photons per decay, which is here experimentally determined to be similar to 20 000. In contrast to the hulk lifetime distributions, the SM fluorescence lifetime distributions exh ibit standard deviations that are sizably larger than the statistically-exp ected values. This comparison proves the strong influence of the inhomogenu ous microenvironment on the photophysical behavior of single molecules embe dded in a 10-30-nm thin polymer layer.