文摘
Two procedures based on the weighted least-squares (LS)and the maximum likelihood estimation (MLE) methodto confidently analyze single-molecule (SM) fluorescencedecays with a total number (N) of 2500-60 000 countshave been elucidated and experimentally compared byanalyzing measured bulk and SM decays. The key observation of this comparison is that the LS systematicallyunderestimates the fluorescence lifetimes by ~5%, for therange of 1000-20 000 events, whereas the MLE methodgives stable results over the whole intensity range, evenat counts N less than 1000, where the LS analysis deliversunreasonable values. This difference can be attributed tothe different statistics approaches and results from improper weighting of the LS method. As expected fromtheory, the results of both methods become equivalentabove a certain threshold of N detected photons perdecay, which is here experimentally determined to be~20 000. In contrast to the bulk lifetime distributions,the SM fluorescence lifetime distributions exhibit standard deviations that are sizably larger than the statisticallyexpected values. This comparison proves the stronginfluence of the inhomogenuous microenvironment on thephotophysical behavior of single molecules embedded ina 10-30-nm thin polymer layer.