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. 2012 Jul 3;103(1):L7-9.
doi: 10.1016/j.bpj.2012.05.038.

NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy

Belinda K Wright, Laura M Andrews, Julie Markham, Mark R Jones, Chiara Stringari, Michelle A Digman, Enrico Gratton

NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy

Belinda K Wright et al. Biophys J. .

Abstract

NADH is a naturally fluorescent metabolite associated with cellular respiration. Exploiting the different fluorescence lifetime of free and bound NADH has the potential to quantify the relative amount of bound and free NADH, enhancing understanding of cellular processes including apoptosis, cancer pathology, and enzyme kinetics. We use the phasor-fluorescence lifetime image microscopy approach to spatially map NADH in both the free and bound forms of live undifferentiated and differentiated myoblast cells. The phasor approach graphically depicts the change in lifetime at a pixel level without the requirement for fitting the decay. Comparison of the spatial distribution of NADH in the nucleus of cells induced to differentiate through serum starvation and undifferentiated cells show differing distributions of bound and free NADH. Undifferentiated cells displayed a short lifetime indicative of free NADH in the nucleus and a longer lifetime attributed to the presence of bound NADH outside of the nucleus. Differentiating cells displayed redistribution of free NADH with decreased relative concentration of free NADH within the nucleus whereas the majority of NADH was found in the cytoplasm.

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Figures

Figure 1
Figure 1
Phasor positions of bound and free NADH obtained from cuvette measurements. (Green line) Maximum and minimum range of relative fraction of bound and free found in the myoblast cells. (Red to blue) Relative amount of bound and free NADH, respectively.
Figure 2
Figure 2
(A and B) Images of undifferentiated and early differentiated L6 rat myoblast, respectively. Scale bar is 5 μm. (C and D) Color-coded FLIM images of A and B, respectively, according to the color scale in Fig. 1. (E and F) Phasor plot histograms of C and D, respectively. The G and S coordinates are the cosine and sine transform respectively, as shown in the Materials and Methods section.
Figure 3
Figure 3
Scatter analysis of 40 undifferentiated myoblast cells and 47 early differentiated cells. The average phasor coordinates are shown for the nuclear and the cytoplasmic region of both types of cells.
See this image and copyright information in PMC

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