Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomography

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.

[1]  Christian Pinali,et al.  Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy , 2011, Nature Protocols.

[2]  C. Sasakawa,et al.  Roles of Autophagy in Elimination of Intracellular Bacterial Pathogens , 2013, Front. Immunol..

[3]  Jun Liu,et al.  Evidence for Pore Formation in Host Cell Membranes by ESX-1-Secreted ESAT-6 and Its Role in Mycobacterium marinum Escape from the Vacuole , 2008, Infection and Immunity.

[4]  David G. Russell,et al.  Mycobacterium and the coat of many lipids , 2002, The Journal of cell biology.

[5]  Harald F Hess,et al.  Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes , 2012, Proceedings of the National Academy of Sciences.

[6]  E. V. van Donselaar,et al.  Gridded Aclar: preparation methods and use for correlative light and electron microscopy of cell monolayers, by TEM and FIB–SEM , 2010, Journal of microscopy.

[7]  M. N. Lebbink,et al.  Tomography of insulating biological and geological materials using focused ion beam (FIB) sectioning and low‐kV BSE imaging , 2009, Journal of microscopy.

[8]  DJ Klinonsky.  Guidelines for the use and interpretation of assays for monitoring autophagy (3rd Ed.) , 2016 .

[9]  B. Humbel,et al.  Correlative light and electron microscopy using immunolabeled resin sections. , 2007, Methods in molecular biology.

[10]  C. Daley,et al.  The pharmacokinetics and pharmacodynamics of pulmonary Mycobacterium avium complex disease treatment. , 2012, American journal of respiratory and critical care medicine.

[11]  Mark Fricker,et al.  Interphase Nuclei of Many Mammalian Cell Types Contain Deep, Dynamic, Tubular Membrane-bound Invaginations of the Nuclear Envelope , 1997, The Journal of cell biology.

[12]  Bradley C. Lowekamp,et al.  3D visualization of HIV transfer at the virological synapse between dendritic cells and T cells , 2010, Proceedings of the National Academy of Sciences.

[13]  P. Peters,et al.  ESX‐1‐mediated translocation to the cytosol controls virulence of mycobacteria , 2012, Cellular microbiology.

[14]  S. Subramaniam,et al.  Site-specific 3D imaging of cells and tissues with a dual beam microscope. , 2006, Journal of structural biology.

[15]  R. Bunge,et al.  Studies on the transplantation of spinal cord tissue in the rat. I. The development of a culture system for hemisections of embryonic spinal cord. , 1973, Brain research.

[16]  O. Kwon,et al.  Nontuberculous Mycobacterial Lung Disease , 2006 .

[17]  John A.G. Briggs,et al.  Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision , 2011, The Journal of cell biology.

[18]  A. G. Monteith,et al.  Imaging Transient Blood Vessel Fusion Events in Zebrafish by Correlative Volume Electron Microscopy , 2009, PloS one.

[19]  V. A. Kelley,et al.  Mycobacterium's arrest of phagosome maturation in macrophages requires Rab5 activity and accessibility to iron. , 2003, Molecular biology of the cell.

[20]  H. Mollenhauer.  Artifacts caused by dehydration and epoxy embedding in transmission electron microscopy , 1993, Microscopy research and technique.

[21]  S. Hell.  Far-Field Optical Nanoscopy , 2007, Science.

[22]  V. Deretic,et al.  Autophagy Is a Defense Mechanism Inhibiting BCG and Mycobacterium tuberculosis Survival in Infected Macrophages , 2004, Cell.

[23]  B. Humbel,et al.  Correlative light and electron microscopy using immunolabeled resin sections. , 2007, Methods in molecular biology.

[24]  Judith Klumperman,et al.  Correlative light-electron microscopy (CLEM) combining live-cell imaging and immunolabeling of ultrathin cryosections , 2008, Nature Methods.

[25]  C. de Chastellier,et al.  Cholesterol depletion in Mycobacterium avium‐infected macrophages overcomes the block in phagosome maturation and leads to the reversible sequestration of viable mycobacteria in phagolysosome‐derived autophagic vacuoles , 2006, Cellular microbiology.

[26]  Peter J. Peters,et al.  M. tuberculosis and M. leprae Translocate from the Phagolysosome to the Cytosol in Myeloid Cells , 2007, Cell.

[27]  Robert Clarke,et al.  Guidelines for the use and interpretation of assays for monitoring autophagy , 2012 .

[28]  J. Cox,et al.  Extracellular M. tuberculosis DNA Targets Bacteria for Autophagy by Activating the Host DNA-Sensing Pathway , 2012, Cell.

[29]  P. Andersen,et al.  The success and failure of BCG — implications for a novel tuberculosis vaccine , 2005, Nature Reviews Microbiology.

[30]  Jocelyn Laporte,et al.  From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy , 2010, PloS one.

[31]  G. Knott,et al.  Serial Section Scanning Electron Microscopy of Adult Brain Tissue Using Focused Ion Beam Milling , 2008, The Journal of Neuroscience.

[32]  N. Hibler,et al.  Arrest of Mycobacterial Phagosome Maturation Is Caused by a Block in Vesicle Fusion between Stages Controlled by rab5 and rab7* , 1997, The Journal of Biological Chemistry.

[33]  E. V. van Donselaar,et al.  Aclar discs: a versatile substrate for routine high‐pressure freezing of mammalian cell monolayers , 2006, Journal of microscopy.

[34]  J. S. Collins,et al.  Spectral properties of fluorescence induced by glutaraldehyde fixation. , 1981, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[35]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[36]  Bradley C. Lowekamp,et al.  Multi-resolution correlative focused ion beam scanning electron microscopy: applications to cell biology. , 2014, Journal of structural biology.

[37]  L. Young,et al.  Nontuberculous Mycobacterial Infections: A Clinical Review , 2004, Infection.

[38]  S. Takeshita,et al.  Mycobacterium marinum Escapes from Phagosomes and Is Propelled by Actin-based Motility , 2003, The Journal of experimental medicine.

[39]  Sriram Subramaniam,et al.  Correlative 3D imaging of whole mammalian cells with light and electron microscopy. , 2011, Journal of structural biology.

[40]  Jeremy N. Skepper,et al.  Biological specimen preparation for transmission electron microscopy , 1999 .

[41]  Florian Beck,et al.  Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography. , 2007, Journal of structural biology.

[42]  Stefan W. Hell,et al.  Protein localization in electron micrographs using fluorescence nanoscopy , 2010, Nature Methods.

[43]  G LoweDavid,et al.  Distinctive Image Features from Scale-Invariant Keypoints , 2004 .

[44]  D. Gusnard,et al.  Cell and organelle shrinkage during preparation for scanning electron microscopy: effects of fixation, dehydration and critical point drying , 1977, Journal of microscopy.

[45]  A. Meijer,et al.  Correlative light and electron microscopy imaging of autophagy in a zebrafish infection model , 2014, Autophagy.

[46]  Michael Unser,et al.  A pyramid approach to subpixel registration based on intensity , 1998, IEEE Trans. Image Process..

[47]  D. Vaux,et al.  The nucleoplasmic reticulum: form and function. , 2011, Trends in cell biology.

[48]  Donald Bliss,et al.  Ion-Abrasion Scanning Electron Microscopy Reveals Surface-Connected Tubular Conduits in HIV-Infected Macrophages , 2009, PLoS pathogens.

[49]  B. Humbel,et al.  Integrated fluorescence and transmission electron microscopy. , 2008, Journal of structural biology.

[50]  Jeffrey Caplan,et al.  High-resolution three-dimensional reconstruction of a whole yeast cell using focused-ion beam scanning electron microscopy. , 2012, BioTechniques.

[51]  Jean Gruenberg,et al.  The biogenesis of multivesicular endosomes , 2004, Nature Reviews Molecular Cell Biology.

[52]  S. Akira,et al.  Intracellular Mycobacterium avium intersect transferrin in the Rab11(+) recycling endocytic pathway and avoid lipocalin 2 trafficking to the lysosomal pathway. , 2010, The Journal of infectious diseases.

[53]  Javier DeFelipe,et al.  Counting Synapses Using FIB/SEM Microscopy: A True Revolution for Ultrastructural Volume Reconstruction , 2009, Front. Neuroanat..

[54]  R. E. Kingsley,et al.  Preparation of cultured mammalian cells for transmission and scanning electron microscopy using Aclar film. , 1988, Journal of electron microscopy technique.