Current label-based methods used to describe cell phenotype have to date proven inadequate for accurately predicting the differentiation potential of many stem cell populations. A technique to detect the potential of NPSCs to generate either neurons or glia would improve the use of these cells in therapies.
The ability to direct differentiation of NPSCs towards neurons and to identify neuron-restricted progenitor cells may provide new therapeutic avenues for stroke, spinal cord injury and age-related cognitive conditions, such as Alzheimer’s and Parkinson’s diseases, which cause loss of neurons. The mammalian brain contains a population of neural stem cells (NSCs), which can self renew and differentiate to give raise to neurons, astrocytes and oligodendrocytes. They are relatively quiescent in adults 
, entering the cell cycle to produce more rapidly dividing progenitors that undergo limited rounds of proliferation and are more committed to specific neural lineages 
. Astrocytes perform many different functions, including providing structural and nutrient support for neurons, secreting signaling molecules, and uptake and metabolism of neurotransmitters.
Traditional cell sorting is performed by flow cytometry or fluorescence-activated cell sorting (FACS) that provide separation of cellular populations based on fluorescent labeling of cell surface markers 
. For example different surface markers have been identified for NSPCs (CD133, SSEA-1 [CD15], A2B5), and differentiated neurons (CD24, NCAM, CD56) 
. Although cell sorting efficiency has been optimized in the last years 
cell viability after sorting is still not very high and the capability of sorted cell further differentiation could be altered. Since these techniques rely on the availability of a marker, the absence of a surface marker that can separate NSPCs with different differentiation fate renders the purification of subset of cells for therapy impossible.
A live-cell label-free measure of fate potential would solve this problem by reducing the need for specific cell surface markers. Label-free techniques are becoming increasingly more popular for their non-invasive features. Some label free techniques have been developed to identify stem cells from their differentiated progenies based on dielectric properties of stem cells 
or chemical analysis by Raman spectroscopy 
Emerging evidence suggests that energy metabolism and the redox state are important regulators of stem cell functions such as self-renewal, differentiation, lineage-specification and stem cell fate options 
. Stem cells possess metabolic characteristics that differ from differentiated cells 
. In the brain, unique features of neurons (e.g. electrical excitability and neurotransmission), oligodendrocytes (e.g. high lipid levels), and astrocytes (e.g. recycling of neurotransmitters and metabolites) also suggest that metabolic requirements of differentiated cells may drastically differ from that of self-renewing, multipotent NSCs. Gene expression analyses have revealed that from development through adulthood, the transition from a NSC/neural progenitor cell to a differentiated neuron, astrocyte, or oligodendrocyte is associated with numerous transcriptional changes, including genes associated with metabolism and energy sensing 
. Cultured postnatal NSCs also show significantly higher expression of numerous metabolic genes 
. Noble et al. 
first observed changes in the intracellular redox state during the self renewal and the differentiation processes of dividing progenitor cells. Only recently a mechanism involved in neuronal differentiation has been identified as requiring SIRT1 activity, which is regulated by nicotinamide adenine dinucleotide (NAD+) and therefore is sensitive to redox state and cell metabolism 
. Prozorovski et al. 
showed that redox state does affect the cell-fate decision of NPCs in vitro
, with oxidizing conditions favoring differentiation into astrocytes, whereas reducing conditions favor neuron formation.
The metabolic coenzyme nicotinamide adenine dinucleotide (NADH) is the principal electron acceptor in glycolysis and electron donor in oxidative phosphorylation. NADH ubiquity renders this coenzyme one of the most useful and informative intrinsic biomarkers for metabolism in live cells and tissues 
. Since the pioneering work of Britton Chance 
metabolic imaging of NADH fluorescence levels and of the relative amounts of reduced and oxidized NADH is extensively used to monitor changes in metabolism. NADH has either a short or long fluorescence lifetime component depending whether it is in a free or protein-bound state. Protein-bound NADH is characterized by a complex multi-exponential lifetime decay that has been related to its binding to different enzymes, such as malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) 
. Metabolic pathways related to carcinogenesis and differentiation are known to change NADH binding sites and enzymatic binding is directly related to NADH cycling through the energy production pathway 
. For example, Bird et al. 2005 showed that changes in the ratio of free to protein-bound NADH are associated with the NADH/NAD+ redox ratio in breast cancer cells 
. NADH has been recently used to discriminate different redox ratios of undifferentiated stem cells and their differentiating progenies 
. We demonstrated that the Phasor approach to fluorescence lifetime microscopy (FLIM) combined with Multi Photon Microscopy (MPM) is a label free and very sensitive method to identify and distinguish different NADH metabolic and differentiation state of germ cells in a living tissue 
Here we use the phasor approach to FLIM to measure and identify the metabolic signature of differentiated neurons and NPSCs at different developmental stages. We find that the ratio of free to protein-bound NADH strongly correlates with the differentiation state of the cells and to their fate commitment. Undifferentiated NPSCs have a glycolytic phenotype characterized by high free/bound NADH, while differentiated neurons have an oxidative phosphorylation phenotype, characterized by low free/bound NADH. Here we show that by measuring the metabolic activity and redox ratio of cells we can distinguish neuronal-biased progenitors from glial-biased progenitors. For the first time we demonstrate that NPSCs committed to different differentiation potentials can be identified by their NADH metabolic states even if they are indistinguishable morphologically and by the expression of lineage markers.