Three-dimensional in situ morphometrics of Mycobacterium tuberculosis infection within lesions by optical mesoscopy and novel acid-fast staining

Francis, Robert J. and Robb, Gillian and McCann, Lee and Khatri, Bhagwati and Keeble, James and Dagg, Belinda and Amos, Brad and Salguero, Francisco J. and Ho, Mei Mei and Bullen, Anwen and McConnell, Gail and MacLellan-Gibson, Kirsty (2020) Three-dimensional in situ morphometrics of Mycobacterium tuberculosis infection within lesions by optical mesoscopy and novel acid-fast staining. Scientific Reports, 10. 21774. ISSN 2045-2322

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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.

    ORCID iDs

    Francis, Robert J., Robb, Gillian ORCID logoORCID: https://orcid.org/0000-0002-3004-5926, McCann, Lee, Khatri, Bhagwati, Keeble, James, Dagg, Belinda, Amos, Brad, Salguero, Francisco J., Ho, Mei Mei, Bullen, Anwen, McConnell, Gail ORCID logoORCID: https://orcid.org/0000-0002-7213-0686 and MacLellan-Gibson, Kirsty;