Differential levels of metabolic activity in isolated versus confluent/partially confluent HeLa cells are analyzed by autofluorescent NAD(P)H using multi-photon FLIM microscopy

NAD(P)H is a known biomarker for cellular metabolism; a higher ratio of enzyme-bound NAD(P)H to free/unbound NAD(P)H indicates an increase in metabolic activity. Free NADH has a shorter fluorescence lifetime (τ 1 ), the bound version (τ 2 ) a longer lifetime. FLIM’s unique capability to establish inter alia the relative fractions of τ 1 (a 1 %) and τ 2 (a 2 %) in each pixel, determines the level of metabolic activity. The relative abundances of bound NAD(P)H were analyzed for single cells, confluent and partially confluent cells within 3 Fields-of-View (FoVs). A gradient of increasing a 2% levels of bound NAD(P)H from single, partially confluent to confluent cells was observed.