Figure 4

Morphometric analysis of infection within lesions. The steps of the image analysis pipeline are presented (A–G). To begin the Mesoscope image (Ai) of a H37Rv infected lung sample, with a zoomed-in image (Aii) showing the Mtb infection as a speckly pattern through the tissue, is mathematically squared (B) and the background subtracted (Ci). A zoomed-in image (Cii) of the yellow region of interest in (Ci) shows the Mtb pattern that has been isolated. A region of interest is then calculated to cover the lesions only (D) and applied to the isolated Mtb signal (C). The individual Z slices are then constructed into a 3D stack, with a maximum intensity projection of this sample shown in (E) and a zoomed-in image at a tilt shown in (F). The average RenyiEntropy threshold calculated across Z Stack and Isosurfaces generated (G) with the volume (µm³) colour coded for visualisation. Examples of morphometric data include total number of volumes (Hi), volume of infection within the granuloma ROIs (Hii), colony volume (Hiii), Surface area (Hiv) and Sphericity (Hv) in H37Rv and Kenyan isolates infected tissue with volume (individual mice shown with n ≥ 500 volumes measured per stack) (Black = Kenyan 1521, Orange = Kenyan 3870, Pink = Kenyan 3894, Grey = Mtb strain H37Rv; Solid colour = BALB/c host strain and Patterned colour = F1 mice). Scale bar = 20 µm (Aii, Cii) and Scale bar = 500 µm (Ai, B, Ci, D, E) unless otherwise presented.