Abstract. Measurement of endogenous free and bound NAD(P)H relative concentrations in living cells is
a useful method for monitoring aspects of cellular metabolism, because the NADH?NAD? reduction-oxidation
pair is crucial for electron transfer through the mitochondrial electron transport chain. Variations of free and
bound NAD(P)H ratio are also implicated in cellular bioenergetic and biosynthetic metabolic changes accompanying
cancer. This study uses two-photon fluorescence lifetime imaging microscopy (FLIM) to investigate
metabolic changes in MCF10A premalignant breast cancer cells treated with a range of glycolysis inhibitors:
namely, 2 deoxy-D-glucose, oxythiamine, lonidamine, and 4-(chloromethyl) benzoyl chloride, as well as the
mitochondrial membrane uncoupling agent carbonyl cyanide m-chlorophenylhydrazone. Through systematic
analysis of FLIM data from control and treated cancer cells, we observed that all glycolytic inhibitors apart
from lonidamine had a slightly decreased metabolic rate and that the presence of serum in the culture medium
generally marginally protected cells from the effect of inhibitors. Direct production of glycolytic L-lactate was also
measured in both treated and control cells. The combination of these two techniques gave valuable insights into
cell metabolism and indicated that FLIM was more sensitive than traditional biochemical methods, as it directly
measured metabolic changes within cells as compared to quantification of lactate secreted by metabolically
active cells.