Figure 4. Autofluorescence imaging resolves temporal changes in the metabolic activity of macrophages at sterile tail wounds.

Tail fin transection (Tt) or thermal injury (burn) distal to the notochord was performed using transgenic zebrafish larvae (Tg(mpeg1:mCherry-CAAX) that labels macrophages in the plasma membrane with mCherry) at 3 days post fertilization. Autofluorescence imaging of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) was performed on live larvae at 24 and 72 hr post wound (Figure 1A). (A) Representative images of mCherry expression to show macrophages, optical redox ratio, and NAD(P)H and FAD mean lifetimes (${\displaystyle {\tau }_m}$) are shown for Tt or burn wounds; macrophages were outlined with dashed lines and the area was overlaid in the optical redox ratio and lifetime images to show corresponding location of macrophages; scale bar = 50 µm. Quantitative analysis of (B) optical redox ratio, (C) Optical Metabolic Imaging index, and (D) NAD(P)H mean lifetime (${\displaystyle {\tau }_m}$) from three biological repeats (Tt-24 hr = 322 cells/16 larvae, burn-24 hr = 850 cells/14 larvae, Tt-72 hr = 213 cells/12 larvae, burn-72 hr = 578 cells/11 larvae) is shown; quantitative analysis of associated NAD(P)H and FAD mean (${\displaystyle {\tau }_m}$) and individual lifetime endpoints (${\displaystyle {\tau }_1,{\tau }_2,{\alpha }_1}$), and sample size for each repeat are included in Figure 4—figure supplement 1. Interaction between treatment and time was included to analyze whether either factor modified the effect of the other; strong interaction was detected for the optical redox ratio. p values represent statistical analysis of the overall effects. Estimated means with 95% CI and overall effects with p values are included in Figure 4—source data 1. (E) Tail fin tissue was collected distal to the caudal vein/artery loop (blue box) 24 hr following either tail transection or thermal injury distal to the notochord (black line) for mass spec analysis of small metabolites to compare the global trend of changes in redox metabolites with that measured by autofluorescence imaging; metabolomics data shown in (E) and (F) are from four biological repeats. (F) Metabolite abundance measured by either autofluorescence imaging or mass spec in transection sample was normalized by that in burn or (G) was used to calculate the redox ratio in transection (Tt) or burn samples. We included NADPH abundance in the redox ratio calculated using mass spec measurements. *NADPH and NADH intensities could not be collected separately by autofluorescence imaging as their fluorescence spectra overlap, thereby were measured collectively.

Figure 4—figure supplement 1. Individual nicotinamide adenine dinucleotide (phosphate) NAD(P)H and flavin adenine dinucleotide (FAD) fluorescence lifetime endpoints associated with Figure 4.

Quantitative analysis of (A) tau1 (${\displaystyle {\tau }_1}$), free/short lifetime of NAD(P)H, (B) tau2 (${\displaystyle {\tau }_2}$), bound/long lifetime of NAD(P)H, (C) alpha1 (α₁), fractional component of free NAD(P)H, (D) mean lifetime (${\displaystyle {\tau }_m}$) of FAD, (E) tau1 (${\displaystyle {\tau }_1}$), bound/short lifetime of FAD, (F) tau2 (${\displaystyle {\tau }_2}$), free/long lifetime of FAD, and (G) alpha1 (α₁), fractional component of bound FAD. Log transformation was applied to ${\displaystyle {\tau }_m,{\tau }_1}$ and ${\displaystyle {\tau }_2}$ prior to analysis. Interaction between treatment and time was included to analyze whether either factor modified the effect of the other; weak interaction was detected for NAD(P)H ${\displaystyle {\tau }_1}$. p values represent statistical analysis of the overall effects. Estimated means with 95% CI and overall effects with p values are included in Figure 4—source data 1. (H) Sample size of data set shown in Figure 4 and this supplement.