Green fluorescent protein emission obscures metabolic fluorescent lifetime imaging of NAD(P)H

Autofluorescence of endogenous molecules can provide valuable insights in both basic research and clinical applications. One such technique is fluorescence lifetime imaging (FLIM) of NAD(P)H, which serves as a correlate of glycolysis and electron transport chain rates in metabolically active tissue. A powerful advantage of NAD(P)H-FLIM is the ability to measure cell-specific metabolism within heterogeneous tissues. Cell-type specific identification is most commonly achieved with directed green fluorescent protein (GFP) expression. However, we demonstrate that NAD(P)H-FLIM should not be analyzed in GFP-expressing cells, as GFP molecules themselves emit photons in the blue spectrum with short fluorescence lifetimes when imaged using two-photon excitation at 750 nm. This is substantially different from the reported GFP emission wavelength and lifetime after two-photon excitation at 910 nm. These blue GFP photons are indistinguishable from free NAD(P)H by both emission spectra and fluorescence lifetime. Therefore, NAD(P)H-FLIM in GFP-expressing cells will lead to incorrect interpretations of metabolic rates, and thus, these techniques should not be combined.