Figure 5
- ID
- ZDB-FIG-220302-228
- Publication
- Miskolci et al., 2022 - In vivo fluorescence lifetime imaging of macrophage intracellular metabolism during wound responses in zebrafish
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(A, B) Thermal injury (burn) distal to the notochord was performed using double transgenic zebrafish larvae (Tg(tnf:GFP x mpeg1:mCherry-CAAX) that labels macrophages in the plasma membrane with mCherry, and monitors TNF? expression using GFP-based TNF? reporter) at 3 days post fertilization (dpf), that were either untreated (control) or treated with 1 mM metformin starting at 1 dpf; larvae were fixed at 24 hr post burn (hpb). (A) Representative images of sum projections of z-stacks acquired by spinning disk confocal microscopy are shown; scale bar = 100 ?m. (B) TNF? expression in macrophages were quantified by area thresholding of GFP and mCherry intensities, and is displayed as proportional area per larva with bars showing arithmetic mean and 95% CI; results are from three biological repeats (control = 42, metformin = 46 larvae). (C?F) Thermal injury distal to the notochord was performed using control or metformin-treated transgenic zebrafish larvae (Tg(mpeg1:mCherry-CAAX)) at 3 dpf. Autofluorescence imaging of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) was performed on live larvae at 24 hpb. (C) Representative images of mCherry expression to show macrophages, optical redox ratio, and NAD(P)H and FAD mean lifetimes () are shown; scale bar = 50 µm. Quantitative analysis of (D) optical redox ratio, (E) Optical Metabolic Imaging index, and (F) NAD(P)H mean lifetime () from three biological repeats (control = 632 cells/13 larvae, metformin = 670 cells/14 larvae) is shown; quantitative analysis of associated NAD(P)H and FAD mean () and individual lifetime endpoints (), and sample size for each repeat are included in Figure 5?figure supplement 1. Log transformation was applied to optical redox ratio prior to analysis. p values represent statistical analysis of the overall effects. Estimated means with 95% CI and overall effects with p values are included in Figure 5?source data 1.
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