• confocal microscopy
• flim
• metabolism
• nadh
• ncx-nes
• neural differentiation
• phasor
• stem cells

Neurogenesis in vivo is a long and complex process, characterised by the successive generation of several distinct cell typesfrom a primordial pluripotent population, neuroepithelial stem (NES) cells. The challenge of recapitulating this phenomenon in vitro has led to the establishment of several cell models that can be used to study it and its alterations, which lead to a wealth of neurological and psychiatric disorders. NCX-NES cells are one of the most recent of such models, and we decided here to employ it to study the changes that take place in cell metabolism during differentiation. In particular, we decided to use a label-free imaging technique, NAD(P)H fluorescence lifetime imaging microscopy (FLIM), followed by phasor analysis. This technique allows to probe the state of NAD(P)H, a common cofactor of metabolic enzymes, and determine whether it is mostly bound to these enzymes or not, which can be in turn traced back to a high/low oxydative activity. The technique is fast, non-destructive and highly sensitive, enabling single-cell and even subcellular analysis. The results tend to support the current opinion of aerobic glycolysis being the main actor at the NES stage but being later superseded by oxydative phosphorylation in mature neurons.