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Updated: Apr 28, 2026
Autofluorescence Imaging to Evaluate Cellular Metabolism
Published on: November 15, 2021
Separating NADH and NADPH fluorescence in live cells and tissues using FLIM
Thomas S Blacker1, Zoe F Mann2, Jonathan E Gale2
11] Centre for Mathematics and Physics in the Life Sciences and Experimental Biology, University College London, London WC1E 6BT, UK [2] Research Department of Cell & Developmental Biology, University College London, London WC1E 6BT, UK [3] Department of Physics and Astronomy, University College London, London WC1E 6BT, UK.
Fluorescence lifetime imaging (FLIM) can now distinguish between NAD(P)H cofactors, revealing distinct metabolic roles in cells. This advance helps quantify cellular energy metabolism and antioxidant defense in conditions like cancer.
Area of Science:
- Cellular metabolism
- Biochemistry
- Molecular imaging
Background:
- Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, are crucial for cellular energy metabolism, biosynthesis, and antioxidant defense.
- The reduced forms, NADH and NADPH, are spectrally identical, hindering their individual quantification in living cells.
Purpose of the Study:
- To develop a method for quantitatively distinguishing between NADH and NADPH in living cells.
- To investigate the metabolic implications of NAD(P)H balance shifts in cellular states, including cancer.
Main Methods:
- Utilized genetic and pharmacological approaches to perturb NAD(P)H metabolism.
- Employed fluorescence lifetime imaging (FLIM) to differentiate between NADH and NADPH based on their distinct fluorescence decay rates.
- Developed a mathematical model to quantify relative NADH and NADPH levels in complex tissues.
Main Results:
- FLIM successfully differentiated quantitatively between NADH and NADPH, overcoming spectral limitations.
- Cellular metabolic states (oxidative vs. glycolytic) did not directly alter NAD(P)H fluorescence decay rates.
- Observed lifetime changes in cancer cells correlated with shifts in the NADPH/NADH balance.
- Identified NADPH-enriched cell populations within the mammalian cochlea, suggesting specialized metabolic functions.
Conclusions:
- FLIM is a powerful tool for distinguishing and quantifying NADH and NADPH in vivo.
- NADPH/NADH balance is a significant indicator of cellular metabolic state, particularly in cancer.
- The discovery of NADPH-enriched cells in the cochlea opens new avenues for understanding tissue-specific metabolic roles.
