Fluorescence lifetime imaging microscopy (FLIM) is a sensitive technique in monitoring functional and conformational states of nicotinamide adenine dinucleotide reduced (NADH) and flavin adenine dinucleotide (FAD),main compounds participating in oxidative phosphorylation in cells. In this study, we have applied FLIM to characterize the metabolic changes in HeLa cells upon bacterial infection and made comparison with the results from the cells treated with staurosporine (STS), a well-known apoptosis inducer. The evolving of NADH's average autofluorescence lifetime during the 3 h after infection with enterohemorragic Escherichia coli (EHEC) or STS treatment has been observed. The ratio of the short and the long lifetime components' relative contributions of NADH increases with time, a fact indicating cellular metabolic activity, such as a decrease of oxidative phosphorylation over the course of infection, while opposite dynamics is observed in FAD. Being associated with mitochondria, FAD lifetimes and redox ratio could indicate heterogeneous mitochondrial function, microenvironment with bacterial infection, and further pathway to cell death. The redox ratios for both EHEC-infected and STS-treated HeLa cells have been observed and these observations also indicate possible apoptosis induced by bacterial infection.
Fluorescence lifetime imaging microscopy (FLIM) has been demonstrated as advantageous at discrimination between
free and protein-bound forms of the NADH coenzyme, providing not only with the lifetimes of the both states (shorter τ1
and longer τ2), but also with the relative concentrations of both (fractions α1 and α2 correspondingly). Given the role of
NADH in cellular energetics, NADH FLIM has been applied for the noninvasive characterization of metabolic changes
in a range of pathologies. However, for the discrimination of pathological states, a proper characterization of NADH
fluorescence lifetime dynamics at physiological conditions has to be conducted. We have applied FLIM NADH for the
characterization of metabolic changes during cell culture growth. Our results demonstrate that during the exponential
growth stage there's a well expressed trends of gradual decrease of the free/bound ratio, as measured from the center
from the cell colonies. At the same time the cells at the edges of a colony exhibit higher values of the ratio. Several
possible reasons for the phenomena observed are discussed.
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