Correlated simultaneous fluorescence and phosphorescence lifetime imaging reveals an association between intracellular oxygen tension and metabolic changes in living cells

Abstract

Disruption of cell respiration and metabolic changes accompanies many tissue disorders including neurodegenerative diseases and cancers. Fluorescence lifetime imaging microscopy (FLIM), a non-invasive and information-rich technique, can be invoked to reveal these changes. The method allows for monitoring of fluorescent intrinsic metabolic coenzymes, first of all, NADH (nicotinamide adenine dinucleotide), on the level of single cells and can be applied to living tissue. The ratio between protein-bound and free NADH gives information about the balance between oxidative phosphorylation and glycolysis in cells. There is a correlation between cellular metabolic activity, redox ratio and fluorescence lifetime of NADH. In combination with laser scanning microscopy, the time-correlated single photon counting (TCSPC) technology enables FLIM NADH mapping of the biomedical samples. On the other hand, TCSPC provides quantification of the phosphorescence lifetime of oxygen-sensing molecules. Accordingly, the oxygendependent quenching of phosphorescence of compounds such as transition metal complexes can be employed for evaluation of oxygen partial pressure (pO2) by PLIM (phosphorescence lifetime imaging microscopy). We demonstrate correlated FLIM/PLIM imaging, which provides simultaneous mapping of NADH and oxygen in living cells. Continuous FLIM/PLIM imaging enables to monitor changes in oxygen levels and cell metabolic status dynamically during PDT and provides new opportunities in theranostics.