Abstract
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.
Highlights
We demonstrate the changes observed in TRP lifetime due to the changes in metabolic flux within live human cells
TRP signals were collected with a 3P-fluorescence lifetime imaging (FLIM)-Förster resonance energy transfer (FRET) imaging system using tumorigenic HeLa and nontumorigenic MCF10A cells along with cell perturbation studies by glucose, in space and time
From spectrofluorometer measurements we found that NADH quenched TRP in solution
Summary
We exploit three-photon (3P)-FLIM-FRET to demonstrate the coenzyme-NADH quenching of TRP in live human tumorigenic and nontumorigenic cells. TRP signals were collected with a 3P-FLIM-FRET imaging system using tumorigenic HeLa and nontumorigenic MCF10A cells along with cell perturbation studies by glucose, in space and time.
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