The fluorescence lifetime technique offers an effective way to resolve fluorescent components with overlapping emission spectra. The presence of multiple fluorescent components in biological compounds can hamper their discrimination. The conventional method based on the nonlinear least-squares technique is unable to consistently determine the correct number of fluorescent components in a fluorescence decay profile. This can limit the applications of the fluorescence lifetime technique in biological assays and diagnoses where more than one fluorescent component is typically encountered. We describe the use of an expectation-maximization (EM) method with joint deconvolution to estimate the fluorescence decay parameters, and the Bayesian information criterion (BIC) to accurately determine the number of fluorescent components. A comprehensive simulation and experimental study is carried out to compare the performance and accuracy of the proposed method. The results show that the EM-BIC method is able to accurately identify the correct number of fluorescent components in samples with weakly fluorescing components.
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