Metabolic mapping of cell culture growth by NADH fluorescence lifetime imaging

Fluorescence lifetime imaging microscopy (FLIM) has been demonstrated as advantageous at discriminating 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 a1 and a2 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.