title: Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomography.
abstract: Mycobacteria pose a threat to the world health today, with pathogenic and opportunistic bacteria causing tuberculosis and non-tuberculous disease in large parts of the population. Much is still unknown about the interplay between bacteria and host during infection and disease, and more research is needed to meet the challenge of drug resistance and inefficient vaccines. This work establishes a reliable and reproducible method for performing correlative imaging of human macrophages infected with mycobacteria at an ultra-high resolution and in 3D. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is applied, together with confocal fluorescence microscopy for localization of appropriately infected cells. The method is based on an Aclar poly(chloro-tri-fluoro)ethylene substrate, micropatterned into an advantageous geometry by a simple thermomoulding process. The platform increases the throughput and quality of FIB/SEM tomography analyses, and was successfully applied to detail the intracellular environment of a whole mycobacterium-infected macrophage in 3D.
authors.affiliation: Centre of Molecular Inflammation Research, Norwegian University of Science and Technology (NTNU), Trondheim, Norway; Department of Cancer Research and Molecular Medicine, NTNU, Trondheim, Norway.
authors.forename: Marianne Sandvold
authors.initials: MS
authors.lastname: Beckwith
authors.affiliation: Department of Physics, NTNU, Trondheim, Norway.
authors.forename: Kai Sandvold
authors.initials: KS
authors.lastname: Beckwith
authors.affiliation: Department of Physics, NTNU, Trondheim, Norway.
authors.forename: Pawel
authors.initials: P
authors.lastname: Sikorski
authors.affiliation: Department of Laboratory Medicine, Children's and Women's Health, NTNU, Trondheim, Norway.
authors.forename: Nan Tostrup
authors.initials: NT
authors.lastname: Skogaker
authors.affiliation: Centre of Molecular Inflammation Research, Norwegian University of Science and Technology (NTNU), Trondheim, Norway; Department of Cancer Research and Molecular Medicine, NTNU, Trondheim, Norway.
authors.forename: Trude Helen
authors.initials: TH
authors.lastname: Flo
authors.affiliation: Department of Cancer Research and Molecular Medicine, NTNU, Trondheim, Norway.
authors.forename: Øyvind
authors.initials: Ø
authors.lastname: Halaas
mesh_terms: D055032:Electron Microscope Tomography; D006801:Humans; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional; D007963:Leukocytes, Mononuclear / Q000382:microbiology* / Q000473:pathology* / Q000648:ultrastructure; D008264:Macrophages / Q000382:microbiology / Q000473:pathology / Q000648:ultrastructure; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D009161:Mycobacterium / Q000502:physiology