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Abstract
Tuberculosis (TB) preclinical testing relies on in vivo models including the mouse aerosol challenge model. The only method of determining colony morphometrics of TB infection in a tissue in situ is two-dimensional (2D) histopathology. 2D measurements consider heterogeneity within a single observable section but not above and below, which could contain critical information. Here we describe a novel approach, using optical clearing and a novel staining procedure with confocal microscopy and mesoscopy, for three-dimensional (3D) measurement of TB infection within lesions at sub-cellular resolution over a large field of view. We show TB morphometrics can be determined within lesion pathology, and differences in infection with different strains of Mycobacterium tuberculosis. Mesoscopy combined with the novel CUBIC Acid-Fast (CAF) staining procedure enables a quantitative approach to measure TB infection and allows 3D analysis of infection, providing a framework which could be used in the analysis of TB infection in situ.
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Details
1 National Institute for Biological Standards and Control (NIBSC), Biological Imaging Group, Analytical and Biological Sciences Division, Hertfordshire, UK (GRID:grid.70909.37) (ISNI:0000 0001 2199 6511)
2 SUPA, University of Strathclyde, Department of Physics, Glasgow, UK (GRID:grid.11984.35) (ISNI:0000000121138138)
3 National Institute for Biological Standards and Control (NIBSC), Bacteriology Division, Hertfordshire, UK (GRID:grid.70909.37) (ISNI:0000 0001 2199 6511)
4 Public Health England, Wiltshire, UK (GRID:grid.271308.f) (ISNI:0000 0004 5909 016X)
5 National Institute for Biological Standards and Control (NIBSC), Biological Imaging Group, Analytical and Biological Sciences Division, Hertfordshire, UK (GRID:grid.70909.37) (ISNI:0000 0001 2199 6511); University College London Ear Institute, London, UK (GRID:grid.83440.3b) (ISNI:0000000121901201)