Determination of the metabolic index using the fluorescence lifetime of free and bound nicotinamide adenine dinucleotide using the phasor approach

Abstract

The fluorescence lifetime of nicotinamide adenine dinucleotide (NADH) is commonly used in conjunction with the phasor approach as a molecular biomarker to provide information on cellular metabolism of autofluorescence imaging of cells and tissue. However, in the phasor approach, the bound and free lifetime defining the phasor metabolic trajectory is a subject of debate. The fluorescence lifetime of NADH increases when bound to an enzyme, in contrast to the short multiexponential lifetime displayed by NADH in solution. The extent of fluorescence lifetime increase depends on the enzyme to which NADH is bound. With proper preparation of lactate dehydrogenase (LDH) using oxalic acid (OA) as an allosteric factor, bound NADH to LDH has a lifetime of 3.4 ns and is positioned on the universal semicircle of the phasor plot, inferring a monoexponential lifetime for this species. Surprisingly, measurements in the cellular environments with different metabolic states show a linear trajectory between free NADH at about 0.37 ns and bound NADH at 3.4 ns. These observations support that in a cellular environment, a 3.4 ns value could be used for bound NADH lifetime. The phasor analysis of many cell types shows a linear combination of fractional contributions of free and bound species NADH.

Keywords: FLIM; NADH; TCSPC; autofluorescence; lifetime; phasor.

Figure 1:. Phasor plot for the NADH lifetimes reported in the literature.

Free-NADH is positioned at the universal circle with a short lifetime around 0.4 ns, on the other hand, bound-NADH has reported different lifetimes. Two values are shown in the figure: 1.7 marked by question marks and 3.4. If the lifetime of 1.7ns should be be correct, then the position of the phasor for pixels in a cell should be inside the triangle represented by the black dashed lines. For the full bound-NADH (equilibrium displaced toward to the enzyme-ligand complex shown with the mark LDH-OA in the figure) the position for the phasor distribution should be at the universal circle, considering that should be single exponential. This fact is true for the bound-NADH when the enzyme is incubated with oxalic acid (black circle, _bNADH/LDH-OA), but is not the case for all other lifetimes reported. The experimental position for the ‘others’ _bNADH fall on the line that joins the free and _bNADH (3.4 ns) marked as (experimental position in the figure); this is also true for the experimental data on cell (see Figure 4A). The metabolic trajectory obtained in the cell acquired by our lab is represented as a red arrow. On the other hand, the expected ‘metabolic trajectory’ by the linear combination between free-NADH and the short lifetime bound-NADH should be represented by the dashed green arrow in figure 1. It is interesting to point out that there is no literature (as far as we know) reporting cells with a linear combination between that region in the phasor plot. Instead, most of the values when resolved for 2 components are along the red arrow.

Figure 2.. Collection of fluorescence lifetime decays by the SPC-830 B&H card.

Fluorescence lifetime decay of Coumarin 6 collected for different time windows using SPC-830 B&H card with the gains of 5 and 2 (Black and red lines). The boxes represent the range used for the phasor transformation for the corresponding B&H data collected in the time-domain.

Figure 3.. Phasor trajectories for free and LDH bound NADH obtained using FLIMBox (A) or SPC-830 B&H card acquisition (B, C).

A) Phasor positions calculated using the FLIMBox for free and LDH bound NADH in increasing percentage (4.4%, 44% and 100% of bound NADH). The dashed red line illustrates the line joining the phasor positions of free to fully bound NADH, otherwise termed as the metabolic trajectory. The experimental points are along this line. B) Phasor positions for the same samples as that of (A) calculated from fluorescence lifetime decays obtained using B&H card and a gain of 2 (B&H_G2). Again, the experimental points are along the line combination line. C) Conversion of fluorescence intensity decays measured using B&H card with a gain of 5. The dotted black line (B) and dotted purple line (C) connect the phasor positions from free and fully bound NADH in each corresponding measurements. The experiment points are along a line, but the line shorter lifetime extreme is outside the universal circle and the fraction fo bound is also incorrect. D) The overlap of phasor trajectories obtained with the FLIMBox (red line) and B&H card (black line) with a gain of 5. The trajectories calculated for B&H card gain 2 (purple) is completely different and along the expected line of linear combination.

Figure 4:. Comparison between FLIMBox and SPC-830 B&H card acquisition of cellular autofluorescence in NADH channel.

A) FLIMBox, B) SPC-830 B&H card acquisition with gain of 2 and C) SPC-830 B&H card acquisition with a gain of 5 for cellular autofluorescence in the NADH channel for MEF cells. The top, middle and bottom row in each case show the intensity image, corresponding phasor plot and phasor mapped NADH cell autofluorescence, respectively. Red and cyan cursors are used to select the phasor positions of free and bound NADH (_fNADH and _bNADH), respectively. The dashed line indicates the linear trajectory between the free and bound NADH). The scale bars represent 5 μm. When the SPC-830 B&H is used with a gain of 5, the experimental phasors are not along the line of linear combination. If the metabolic index is calculated for this data set, it has an incorrect value as shown in column C. For this graph the values of bound (3.4ns) and free (0.4) ns for NADH are used, but the conversion to phasor is incorrect so that the phasors points are not in the line of linear combination. In this case, the cell shown in column C) will be misclassified.

Figure 5.

Illustration of the error introduced in the calculation of the metabolic index by NADH FLIM if the lifetime of bound NADH is incorrectly assumed. In this example the metabolic index of cells in a cancer tissue changes toward the glycolytic direction due to the Warburg effect. Along the metabolic trajectory from 3.4ns to 0.4ns the cancer cells are colored and a fraction of free NADH of 0.8 is found. This fraction is intensity weighted. However if the same cells are analyzed using different values for the bound NADH species the position along the metabolic trajectory will move to a fraction of 0.6 for the case of 1.6ns and to 0.83 for the case of 6.5 ns. Cell displaying the Warburg effect will move from (b) to (c) given an erroneous value of the fraction of the bound NADH and the cancer cells could be improperly classified as “normal” for the case of 1.6ns. The point c shows how cancer cell affected by the Warburg effect will be at a different point in the phasor plot, in the case of using 6.5 ns for bound NADH.