Fig. 4

An established model was used in which zebrafish embryos were injected with M. marinum to form localized mycobacterial lesions. All analysis was performed at 4 days post injection. a The model was verified by staining the lesions with a Ziehl–Neelsen stain, highlighting acid-fast mycobacteria (regions of purple), with a representative image selected from three independent biological replicates (N = 3). Scale bar: 20 µm. b Transmission electron micrograph of a mycobacterial lesion, a representative image of two independent biological replicates (N = 2). Osmium staining revealed the presence of mycobacteria (red arrows) with dense lipid clusters (stained black). The plasma membrane of the host cell is also visible (orange arrow). Scale bar: 2 µm. c Confocal Raman spectroscopic imaging (cRSI) was used to image mycobacterial lesions. The image shown is representative of four independent biological replicates (N = 4). Univariate analysis was performed by integrating over a wavenumber range corresponding to relevant biomolecules: protein-rich regions at 2940 ± 16 cm⁻¹ (shown in green), lipid-rich regions at 2854 ± 10 cm⁻¹ (shown in red), DNA-rich regions at 789 ± 10 cm⁻¹ (shown in blue). Scale bars: 40 µm. d Representative spectra obtained from cRSI.