Fluorescence lifetime characterization of bacteria using total lifetime distribution analysis with the maximum entropy method

Total lifetime distribution analysis was employed to obtain fluorescence lifetime profiles of the intrinsic fluorescence ofPseudomonas fluorescens, Escherichia coli, Bacillus subtilis, andStaphylococcus epidermidis. The lifetimes were measured using a multiharmonic Fourier transform phase-modulation fluorometer which can simultaneously measure the phase shift and demodulation at many modulation frequencies. The 364-nm line from an argon-ion laser and the 325- and 442-nm lines from a helium-cadmium laser were used for sample excitation. Broad emission windows were used to capture as much of the bacterial emission as possible for the lifetime measurements. The maximum entropy method was used to recover lifetime profiles from the multifrequency phasemodulation data. At all three excitation wavelengths, the bacteria exhibited three lifetime components, in the ranges of 0.5-1, 2–3, and 4–8 ns. Using 325-nm excitation, a fourth component, in the range of 9–14 ns, was recovered in all of the bacteria; using 364-nm excitation, the fourth component was resolved only in the two Gram-negative bacteria (P. fluorescens andE. coli). Excitation at 364 nm provided the most reproducible lifetime profiles and showed some differences among the four bacteria.