Ultrastructural Imaging of Salmonella-Host Interactions Using Super Resolution Correlative-Light-Electron Microscopy of Bioorthogonal Pathogens
Sander van Kasteren
Bioorthogonal chemistry has been a major breakthrough technique in the study the interaction of this host-pathogen interaction. Through hijacking of the cell wall or protein synthesis biosynthetic machinery, so-called bioorthogonal analogues of cell wall components and amino acids have been incorporated into bacteria. These can be selectively chemically ligated using ‘click’-reactions. This has been particularly exciting for the study of intracellular pathogens as it has allowed their study inside host immune cells, where they hide and replicate. We have recently reported an approach that allows the visualization of these bacteria using correlative light and electron microscopy (CLEM). This technique in which a fluorescence image is overlaid onto an electron micrograph, provides not only their location, but also the whole ultrastructure of the cell in which it resides. One major limitation of this technique has been the resolution of the confocal imaging: whereas the resolution of the EM micrograph is in the order of 1 nm, that of the fluorescence is limited by the Abbe diffraction limit of half the wavelength of the photon (approx. 250 nm). To circumvent this, we have developed an improved approach: by using super-resolution microscopy on the sections, we can now image bioorthogonally labelled pathogens inside phagocytes with optical resolutions of ~20nm, yet with full ultrastructural information on where in the cell the pathogen resides.
Sander van Kasteren obtained his PhD in organic chemistry from the University of Oxford, where – under the tutelage of Prof. Benjamin G. Davis, he worked on the site-selective modification of proteins, as well as on the synthesis of imaging agents for inflammation. Triggered by this application of his compounds in immunological studies, he used his Henry Wellcome Fellowship to do a postdoctoral project in the group of Colin Watts looking at antigen processing and presentation by immune cells leading to the activation of cytotoxic T-cells. His second postdoctoral project in the group of Huib Ovaa took him back to the Netherlands where he took up a an assistant professorship in chemical biology at Leiden University in 2012. In this year he was also awarded the early career research award from the British Biochemical Society, followed by an ERC starting grant in 2014. At present the research in his group is aimed towards understanding the interaction of the immune system with the outside world; especially on the way in which information is transferred during early encounters with pathogens/vaccines to lead to the adaptive immune response. This is important for the development of better vaccines, but also to better understand auto-immune disease emergence.
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