Journal: bioRxiv

Article Title: Correlative Light Electron Ion Microscopy reveal in vivo localisation of bedaquiline in Mycobacterium tuberculosis infected lungs

doi: 10.1101/2020.08.05.237537

Figure Lengend Snippet: (A) Diagram illustrating the in vivo experimental setting. C3HeB/FeJ mice were infected with Mycobacterium tuberculosis H37Rv expressing E2-Crimson (Mtb-E2Crimson) by aerosol. After 21 days, infected mice were treated with 25mg/kg of Bedaquiline (BDQ) or vehicle daily for 5 days via oral gavage. Lungs were removed, fixed with 10% formalin, contrasted and embedded in low melting point agarose then imaged by μCT for sequential vibratome sectioning. (B) Fluorescence microscopy: A tile scan of a tissue section (~100 μm thickness) stained with DAPI (blue) and BODIPY (green), granulomatous lesions are marked with a solid white line to indicate the boundary (scale bar = 1000 μm). (C) Light microscopy of a Region of Interest (ROI): (i) zoomed fluorescence image (white box from ), “landmarks” used for downstream location recognition, are indicated by the solid white boundary lines (scale bar = 500 μm). white rectangle shows the ROI for downstream analysis. (ii) A confocal image of the region indicated by the white box above shows an area of strong cellular infiltration and the accumulation of BODIPY (green) positive cells. Cells infected with Mtb-E2Crimson (red) are also visible throughout this region. The same landmarks marked in image (i) are present. white box indicates the selected infected foamy cell. Scale bar = 100 μm. (iii) Zoomed in image showing the selected foamy cell infected with Mtb-E2Crimson (red) for correlative analysis. Scale bar = 5 μm. (D) Electron microscopy of the ROI: (ii) tissue overview (600x magnification) with landmarks present, white box indicates the selected infected foamy cell. Scale bar = 100 μm. (iii) Zoomed in image showing the selected foamy cell infected with Mtb (15,000x magnification). Scale bar = 5 μm. (E) Ion microscopy of the selected cell: Panel shows the individual nanoSIMS images for the following ion signals from left to right; 12 C 14 N − , 31 P − , 32 S − and 79 Br − . (F) Correlative light, electron and ion microscopy in tissue (CLEIMiT): Left, a correlated image overlaying fluorescent signal from BODIPY (green) and Mtb-E2Crimson (red) against the SEM image. Right, a correlated image overlaying the 79 Br − and 31 P − signals with the SEM image. Center, the corresponding SEM image of the infected foamy cell. Scale bar = 5 μm.

Article Snippet: In a 24-well plate, 100μm lung slices were washed twice in 200 mM HEPES buffer then incubated for 20 min in a staining solution containing: 0.715 μM DAPI (4′,6-diamidino-2-phenylindole) (ThermoFisher Scientific D1306), 10 mg/L BODIPY 493/503 (4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene) (Invitrogen D3922) in 200 mM HEPES buffer.

Techniques: In Vivo, Infection, Expressing, Aerosol, Fluorescence, Microscopy, Staining, Light Microscopy, Electron Microscopy