Biological Imaging Group, Analytical and Biological Sciences Division, National Institute for Biological Standards and Control (NIBSC), South Mimms, Hertfordshire, EN6 3QG, UK.
Department of Physics, SUPA, University of Strathclyde, 107 Rottenrow, Glasgow, G4 0NG, UK.
Sci Rep. 2020 Dec 11;10(1):21774. doi: 10.1038/s41598-020-78640-4.
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.
结核病（TB）临床前检测依赖于体内模型，包括小鼠雾化挑战模型。确定组织内 TB 感染的菌落形态计量的唯一方法是二维（2D）组织病理学。2D 测量考虑了单个可观察切片内的异质性，但不考虑上下部分，因为上下部分可能包含关键信息。在这里，我们描述了一种新方法，使用光学透明化和共聚焦显微镜和体视学的新型染色程序，以亚细胞分辨率在大视场范围内对病变内的 TB 感染进行三维（3D）测量。我们表明可以在病变病理学中确定 TB 形态计量，并确定不同结核分枝杆菌菌株感染的差异。体视学结合新型立方酸快速（CAF）染色程序可以实现定量测量 TB 感染，并允许对感染进行 3D 分析，为原位 TB 感染分析提供了一个框架。
