Fig. 6

Living zebrafish were imaged for the 12 h following a controlled stab wound performed at 3 days post fertilization. a Confocal fluorescence microscopy of F-actin was highlighted by phalloidin staining (red) at 3, 6 and 12 hours post wounding (hpw) on three independent biological replicates at each time point (N = 3). b Univariate analysis of the confocal Raman spectroscopic imaging (cRSI) scans of a living zebrafish embryo at 1–3, 4–6 and 10–12 hpw. Univariate analysis was performed by integrating over a wavenumber range at 1450 ± 30 cm⁻¹. c The tissue damage in these images was clearly visualized by using multivariate vertex component analysis (VCA), with the wounded component displayed in red and the unwounded component displayed in green. A water component (black) and a pigment component (yellow) were also identified. Scale bars: 40 µm. d The three tissue endmembers identified by VCA (the water endmember spectra not shown). e Analysis of the unwounded and wounded endmembers identified by the VCA. The difference spectrum when the wounded endmember was subtracted from the unwounded endmember. The horizontal dashed line indicates zero difference between the two endmembers. The dotted vertical lines indicate the center of the annotated peaks that were clearly different between wounded and unwounded tissue, with the color coding indicating in which component they were dominant. Green indicates higher presence in the unwounded component and red indicates higher presence in the wounded component. f Relative abundance of wounded and unwounded component in the injured somite at the three time points extracted from pixel intensity histograms produced by the VCA. The wounded component is shown in red and the unwounded component is shown in green. Figures 6b–f are a representative set of images and analysis taken from three independent biological replicates (N = 3).