Figure 1.
Free and bound NADH distribution within NPSCs.
Two-photon fluorescence intensity images (a) and free/bound NADH FLIM maps (b) of the E12 NPSCs excited at 740 nm (c) FLIM phasor plot of E12 NPSCs autofluorescence. A linear cluster represents relative concentrations of free NADH (purple) and bound NADH (cyan-white). Red-purple color indicates a high free/bound NADH ratio, while violet, cyan and white indicate linearly and progressively decreasing ratios of free/bound NADH. (d) Scatter plot of the cell mitochondria (blue circle) and cell nuclei (red triangles). Nuclei contain a higher concentration of free NADH, while mitochondria contain mainly bound NADH.
Figure 2.
NADH is the major intrinsic source in NPSCs and neurons.
(a–c) Spectral images and (a’–c’) transmission images of E12 NPSCs (a), E16 NPSCs (a), and neurons excited at 740 nm. (d–f) Emission spectrum measured in the E12 NPSCs (d), E16 NPSCs (e), and neurons (f) respectively.
Figure 3.
Stability of the Phasor FLIM signature of cells.
Two-photon fluorescence intensity images (a) and free/bound NADH FLIM maps (b) of the E12 NPSCs excited at 740 nm for 10–20 frames and 40–50 frames. The same field of view is scanned for a total of 70 frames. (c) FLIM phasor plot of E12 NPSCs autofluorescence. A linear cluster represents relative concentrations of free NADH (purple) and bound NADH (cyan-white). Red-purple color indicates a high free/bound NADH ratio, while violet, cyan and white indicate linearly and progressively decreasing ratios of free/bound NADH. (d) Scatter plot of the average phasor value of two E12 NPSCs that calculated every 10 frames. Numbers indicate the consecutive frames of the scanning. (e) Standard deviations (Ncolony = 6) of the phasor g coordinates of the cell phasor of single E12 NPSCs.
Figure 4.
Reducing and oxidizing conditions shift cellular fingerprint along the M-trajectory from free/bound NADH.
(a–b) Phasor FLIM color maps at 740 nm of the relative concentrations of free NADH (purple) and bound NADH (cyan-white) NADH of E12 NPSCs before and after the addition of potassium cyanide, which blocks the respiration chain (a) and of NIH3T3 fibroblast before and after the addition of Hydrogen Peroxide (H2O2), which induced oxidative stress. (b) (c–d) FLIM phasor plot of E12 NPSCs in reducing condition before and after the addition of KCN (c) and of NIH3T3 cells in oxidative stress conditions before and after the addition of H2O2 (d). Linear cluster represents all possible relative concentrations of free NADH (purple) and bound NADH (white). (e) Schematic diagram indicates that the accumulation of reduced NADH by blocking the respiration chain shifts cellular metabolic signature toward high ratios of free/bound NADH. (f) Schematic diagram indicates that reduction of the fluorescent reduced pool of NADH by oxidative stress induced by hydrogen peroxide shifts cellular metabolic signature toward low ratios of free/bound NADH.
Figure 5.
Glycolysis and oxidative phosphorylation shift cellular fingerprint along the Metabolic trajectory from free to bound NADH.
(a–b) Phasor FLIM color maps at 740 nm of the relative concentrations of free NADH (purple) and bound NADH (cyan-white) NADH of NIH3T3 fibroblast in low glucose (4.5 mM) and high glucose (22 mM) (a) and of control DLD-1 colon cancer cells and DLD-1 colon cancer cells treated with sodium dichloroacetate (DCA). (b) Dichloroacetate ion inhibits pyruvate dehydrogenase kinase, resulting in the inhibition of glycolysis and a decrease in lactate production. (c–d) FLIM phasor plot of NIH3T3 cells in low and high glucose (c) and control cancer cells and cells treated with DCA.(d) Linear cluster represents all possible relative concentrations of free NADH (purple) and bound NADH (white). Phasor FLIM distribution shifts toward free NADH with an increasing concentration of glucose, while the phasor FLIM distribution shifts toward bound NADH with DCA treatment. (e) Schematic diagram indicates that glucose uptake shifts cellular metabolic signature toward a glycolytic phenotype with high ratios of free/bound NADH. (f) Schematic diagram indicates that the inhibition of glycolysis through DCA treatment shifts cellular metabolic signature toward an oxidative phosphorylation phenotype with low ratios of free/bound NADH.
Figure 6.
NPSCs and neurons have a unique NADH metabolic fingerprint which predicts developmental stage.
Two-photon fluorescence intensity images (a) and free/bound NADH FLIM maps (b) of E12 NPSCs, E16 NPSCs and neurons. (c) Phasor FLIM distribution of E12 NPSCs, E16 NPSCs and neurons. A linear cluster represents the relative concentrations of free NADH (purple) and bound NADH (white). Purple color indicates a high free/bound NADH ratio, while violet, cyan and white indicate linearly and progressively decreasing ratios free/bound NADH ratio. (d) Scatter plot of the cell phasor of E12 NPSCs (red triangles), E16 NPSCs (blue circles) and differentiated neurons (green squares). During differentiation the cell phasor shifts toward the longer lifetime indicating a decrease of free/bound NADH ratio. (e) Schematic diagram of the NPSCs differentiation following a metabolic trajectory (M-trajectory) from free to protein-bound NADH, with a shift from a glycolytic phenotype to an oxidative phosphorylation phenotype.
Figure 7.
Metabolic heterogeneity of NSPCs and neurons.
(a) Scatter plot of the cell phasor of E12 NPSCs (red squares), E16 NPSCs (blue squares) and differentiated neurons (green squares) isolated from four different animals. The passage numbers for each experiment are: #1 passage 2, #2 passage 1, #3 passage 7, #4 passage 3. During differentiation the cell phasor shifts toward the longer lifetime indicating a decrease of free/bound NADH ratio. (b) Standard deviations Δg (Nanimals = 4) of the phasor g coordinates of the cell phasor of single E12 NPSCs, E16 NPSCs and neurons.