A method to investigate the metabolic activity of intracellular tryptophan (TRP) and coenzyme-NADH using three-photon (3P) fluorescence lifetime imaging (FLIM) and Förster resonance energy transfer (FRET) is presented. Through systematic analysis of FLIM data from tumorigenic and nontumorigenic cells, a statistically significant decrease in the fluorescence lifetime of TRP was observed in response to the increase in protein-bound NADH as cells were treated with glucose. The results demonstrate the potential use of 3P-FLIM-FRET as a tool for label-free screening of the change in metabolic flux occurring in human diseases or other clinical conditions.
Changes in energy metabolism, mitochondrial functions and of reactive oxygen species are often supposed to induce
alterations in cellular activity. The major intracellular endogenous fluorophores are reduced nicotinamide adenine
dinucleotide (NADH) and dinucleotide phosphate (NADPH), riboflavin's, and tryptophan present inside biological
tissue and they can be used to image tissue architecture without any exogenous probe. Their fluorescence can be excited
by multi-photon microscopy using NIR laser wavelengths [1,2,5,6]. Using FLIM imaging the lifetime of tryptophan and
NADH were monitored in cells with and without addition of glucose in the medium. The lifetime data were collected
and further using the ANOVA (refer table 2) of the lifetime of free NADH, bound NADH and Tryptophan, we found on
applying the null hypothesis for the P-value > 0.05, there is a significant difference between the lifetimes of bound
NADH and Tryptophan from control to glucose treated cells, however, free NADH, shown to be not significant change
between the control and glucose treated cells. The tryptophan puzzle comes closer and closer to a solution. The ultimate
evidence supporting the existence of FRET between Tryptophan and NADH in live cells slowly could come from
lifetime measurements of tryptophan in proteins and bound NADH within live cells.