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Cellular Imaging of Intracellular Bacterial Pathogens

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  • Authors: Virginie Stévenin1, Jost Enninga2
  • Editors: Pascale Cossart3, Craig R. Roy4, Philippe Sansonetti5
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Institut Pasteur, BCI, Paris 75015, France; 2: Institut Pasteur, BCI, Paris 75015, France; 3: Institut Pasteur, Paris, France; 4: Yale University School of Medicine, New Haven, Connecticut; 5: Institut Pasteur, Paris, France
  • Source: microbiolspec April 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.BAI-0017-2019
  • Received 29 August 2018 Accepted 10 January 2019 Published 05 April 2019
  • Jost Enninga, [email protected]
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  • Abstract:

    The spatial dimensions of host cells and bacterial microbes are perfectly suited to being studied by microscopy techniques. Therefore, cellular imaging has been instrumental in uncovering many paradigms of the intracellular lifestyle of microbes. Initially, microscopy was used as a qualitative, descriptive tool. However, with the onset of specific markers and the power of computer-assisted image analysis, imaging can now be used to gather quantitative data on biological processes. This makes imaging a driving force for the study of cellular phenomena. One particular imaging modality stands out, which is based on the physical principles of fluorescence. Fluorescence is highly specific and therefore can be exploited to label biomolecules of choice. It is also very sensitive, making it possible to follow individual molecules with this approach. Also, microscopy hardware has played an important role in putting microscopy in the spotlight for host-pathogen investigations. For example, microscopes have been automated for microscopy-based screenings. A new generation of microscopes and molecular probes are being used to image events below the resolution limit of light. Finally, workflows are being developed to link light microscopy with electron microscopy methods via correlative light electron microscopy. We are witnessing a golden age of cellular imaging in cellular microbiology.

  • Citation: Stévenin V, Enninga J. 2019. Cellular Imaging of Intracellular Bacterial Pathogens. Microbiol Spectrum 7(2):BAI-0017-2019. doi:10.1128/microbiolspec.BAI-0017-2019.

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/content/journal/microbiolspec/10.1128/microbiolspec.BAI-0017-2019
2019-04-05
2019-10-15

Abstract:

The spatial dimensions of host cells and bacterial microbes are perfectly suited to being studied by microscopy techniques. Therefore, cellular imaging has been instrumental in uncovering many paradigms of the intracellular lifestyle of microbes. Initially, microscopy was used as a qualitative, descriptive tool. However, with the onset of specific markers and the power of computer-assisted image analysis, imaging can now be used to gather quantitative data on biological processes. This makes imaging a driving force for the study of cellular phenomena. One particular imaging modality stands out, which is based on the physical principles of fluorescence. Fluorescence is highly specific and therefore can be exploited to label biomolecules of choice. It is also very sensitive, making it possible to follow individual molecules with this approach. Also, microscopy hardware has played an important role in putting microscopy in the spotlight for host-pathogen investigations. For example, microscopes have been automated for microscopy-based screenings. A new generation of microscopes and molecular probes are being used to image events below the resolution limit of light. Finally, workflows are being developed to link light microscopy with electron microscopy methods via correlative light electron microscopy. We are witnessing a golden age of cellular imaging in cellular microbiology.

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Figures

Image of FIGURE 1
FIGURE 1

Wavelengths, objects, and their recognition. The sizes of different objects and the tools that can be used for their visualization are shown.

Source: microbiolspec April 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.BAI-0017-2019
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Image of FIGURE 2
FIGURE 2

The Jablonski diagram explains the basic principles of fluorescence. Molecules are excited by incoming light to reach a higher energy level (S). After vibrational loss of energy (among other losses), the molecule falls back to its low energy level (S), emitting fluorescent light. An excitation-and-emission spectrum for a hypothetical fluorophore that could be similar to GFP is shown on the right. Image courtesy of Gael Moneron (Institut Pasteur).

Source: microbiolspec April 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.BAI-0017-2019
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Image of FIGURE 3
FIGURE 3

Fixed confocal acquisition of ruffle formations and entry in HeLa cells. All salmonellae expressed a fluorescent plasmid (in red). lipopolysaccharide is immunolabeled before cell permeabilization (in green). The host actin cytoskeleton and nuclei are stained with phalloidin (in grey) and DAPI (4′,6-diamidino-2-phenylindole; in blue). Bars, 10 μm.

Source: microbiolspec April 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.BAI-0017-2019
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Image of FIGURE 4
FIGURE 4

Time-lapse imaging of epithelial cells infected with using bacterial and host genetically encoded probes. HeLa cells were transfected with GFP-actin (top) or GFP-Rab5 (bottom) to follow the ruffle formation and the phagosomal trafficking upon infection with dsRed-expressing (yellow arrowheads indicate ruffles). White bars, 10 μm; yellow bars, 2 μm.

Source: microbiolspec April 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.BAI-0017-2019
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Image of FIGURE 5
FIGURE 5

Statistical tools for imaging-based screens. () Statistical analysis tests of knockdown screens to assess quality control. () Statistical analysis tests for data normalization and hit identification from the screens. Mathematical formulas and interpretation are shown. Adapted from references 27 and 28 .

Source: microbiolspec April 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.BAI-0017-2019
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Image of FIGURE 6
FIGURE 6

Large-volume CLEM workflow via multidimensional confocal and FIB/SEM imaging. Biological samples are prepared on gridded glass-bottom slides for time-lapse imaging (1), and events are tracked dynamically at high resolution (2). After site identification under the light microscope (3), locations are retrieved in the electron microscope (4), and three-dimensional volumes are obtained by milling and scanning of the prepared specimen (5). Afterwards, both image data sets are correlated and segmented (6). A typical data set spans 10 μm by 10 μm by 10 μm. Invading organisms are depicted, the forming entry foci are segmented (gold), and macropinosomes (orange) in the vicinity of the entering bacteria (blue) are identified. Images were taken by Allon Weiner (Institut Pasteur).

Source: microbiolspec April 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.BAI-0017-2019
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