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Journal of Biomedical Optics • Open Access

Multiphoton fluorescence lifetime imaging microscopy reveals free-to-bound NADH ratio changes associated with metabolic inhibition
Author(s): Krystyna Drozdowicz-Tomsia; Ayad G. Anwer; Michael A. Cahill; Kaiser N. Madlum; Amel M. Maki; Mark S. Baker; Ewa M. Goldys

Paper 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.

Paper Details

Date Published: 20 August 2014
PDF: 13 pages
J. Biomed. Opt. 19(8) 086016 doi: 10.1117/1.JBO.19.8.086016
Published in: Journal of Biomedical Optics Volume 19, Issue 8
Show Author Affiliations
Krystyna Drozdowicz-Tomsia, Macquarie Univ. (Australia)
Ayad G. Anwer, Macquarie Univ. (Australia)
Michael A. Cahill, Charles Sturt Univ. (Australia)
Kaiser N. Madlum, Univ. of Baghdad (Iraq)
Amel M. Maki, Univ. of Baghdad (Iraq)
Mark S. Baker, Macquarie Univ. (Australia)
Ewa M. Goldys, Macquarie Univ. (Australia)


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