Interpreting NADH fluorescence lifetime across timescales.

NADH fluorescence lifetime imaging can reflect glycolysis–oxidative respiration balance, but its interpretation can fail when NADH-binding landscapes shift slowly over hours to days or rapidly via transient metabolite effects.

• Why it matters: The common assumption that NADH-binding properties stay stable during an experiment is not always valid, risking misinterpretation of metabolic state.
• What they did: This Perspective reviews two failure modes—enzyme identity/abundance changes during prolonged treatments or differentiation, and transient metabolite-driven changes in NADH engagement—then provides guidance for experimental design and interpretation.
• The result: By clarifying when each concern applies, the work helps researchers choose conditions and interpret NADH lifetime data more reliably across experimental timescales.

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