In vivo fluorescence lifetime imaging of macrophage intracellular metabolism during wound responses in zebrafish

Veronika Miskolci, Kelsey E. Tweed, Michael R. Lasarev, Emily C. Britt, Alex J. Walsh, Landon J. Zimmerman, Courtney E. McDougal, Mark R. Cronan, Jing Fan, John Demian Sauer, Melissa C. Skala, Anna Huttenlocher

Research output: Contribution to journalArticlepeer-review

18 Scopus citations


The function of macrophages in vitro is linked to their metabolic rewiring. However, macro-phage metabolism remains poorly characterized in situ. Here, we used two-photon intensity and lifetime imaging of autofluorescent metabolic coenzymes, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD), to assess the metabolism of macrophages in the wound microenvironment. Inhibiting glycolysis reduced NAD(P)H mean lifetime and made the intracellular redox state of macrophages more oxidized, as indicated by reduced optical redox ratio. We found that TNFα+ macrophages had lower NAD(P)H mean lifetime and were more oxidized compared to TNFα− macrophages. Both infection and thermal injury induced a macrophage population with a more oxidized redox state in wounded tissues. Kinetic analysis detected temporal changes in the optical redox ratio during tissue repair, revealing a shift toward a more reduced redox state over time. Metformin reduced TNFα+ wound macrophages, made intracellular redox state more reduced and improved tissue repair. By contrast, depletion of STAT6 increased TNFα+ wound macrophages, made redox state more oxidized and impaired regeneration. Our findings suggest that autofluorescence of NAD(P)H and FAD is sensitive to dynamic changes in intracellular metabolism in tissues and can be used to probe the temporal and spatial regulation of macrophage metabolism during tissue damage and repair.

Original languageEnglish (US)
Article numbere66080
StatePublished - Feb 24 2022


  • Animals
  • Female
  • Flavin-Adenine Dinucleotide/metabolism
  • Fluorescence
  • Glycolysis
  • Kinetics
  • Macrophages/metabolism
  • Mice
  • Mice, Inbred C57BL
  • Microscopy, Fluorescence, Multiphoton/methods
  • NADP/metabolism
  • Oxidation-Reduction
  • Tumor Necrosis Factor-alpha/metabolism
  • Wounds and Injuries/metabolism
  • Zebrafish/metabolism

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)


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