Bioenergetic Alterations of Metabolic Redox Coenzymes as NADH, FAD and FMN by Means of Fluorescence Lifetime Imaging Techniques

Abstract

Metabolic FLIM (fluorescence lifetime imaging) is used to image bioenergetic status in cells and tissue. Whereas an attribution of the fluorescence lifetime of coenzymes as an indicator for cell metabolism is mainly accepted, it is debated whether this is valid for the redox state of cells. In this regard, an innovative algorithm using the lifetime characteristics of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) to calculate the fluorescence lifetime induced redox ratio (FLIRR) has been reported so far. We extended the FLIRR approach and present new results, which includes FLIM data of the various enzymes, such as NAD(P)H, FAD, as well as flavin mononucleotide (FMN). Our algorithm uses a two-exponential fitting procedure for the NAD(P)H autofluorescence and a three-exponential fit of the flavin signal. By extending the FLIRR approach, we introduced FLIRR1 as protein-bound NAD(P)H related to protein-bound FAD, FLIRR2 as protein-bound NAD(P)H related to free (unbound) FAD and FLIRR3 as protein-bound NAD(P)H related to protein-bound FMN. We compared the significance of extended FLIRR to the metabolic index, defined as the ratio of protein-bound NAD(P)H to free NAD(P)H. The statistically significant difference for tumor and normal cells was found to be highest for FLIRR1.

Keywords: FAD; FLIM; FLIRR index; FMN; NAD(P)H; NAD(P)H metabolic index; OXPHOS; cell metabolism; extended FLIRR; glycolysis.

Figure 1

Intracellular coenzymes that play key roles in energy metabolism and metabolic FLIM and their spectral and lifetime characteristics obtained from published data [10,12,13,14,15]. The figure was prepared using the software “ChemDraw (version 20.1.0.110)”.

Figure 2

Intensity preview images obtained during lifetime measurements after two-photon excitation at 880 nm (a) and 780 nm (b) of HaCaT cells. Fluorescence was detected in the spectral range 426–446 nm (green channel) and 542–582 nm (red channel).

Figure 3

Images of the intracellular distribution of the metabolic NAD(P)H index and FLIRR1, FLIRR2, FLIRR3 indices within HaCaT and SCC4 cells. Scale bar 20 µm.

Figure 4

Fluorescence lifetime images of co-cultured HaCaT and SCC4 cells. The autofluorescence of NAD(P)H was recorded in the “green” (426–446 nm) spectral channel under 780 nm two-photon excitation whereas the autofluorescence of flavins was collected in the “red” (542–582 nm) spectral channel under 880 nm two-photon excitation. Scale bar 20 µm.

Figure 5

Intracellular distribution of the metabolic index and the redox ratio FLIRR1 within co-cultured HaCaT and SCC4 cells. Scale bar 20 µm.