A report from the Centre d’immunologie de Marseille-Luminy (CIML, Center of Immunology Marseille-Luminy). From worm to man, from molecules to the whole organism, from physiology to pathology, researchers at the CIML study all areas of immunology.

 

Whoa, is that a creature from outer space? Not exactly. It is from the Centre d’immunologie de Marseille-Luminy (CIML), directed by Philippe Pierre who has taken the helm from Éric Vivier. More specifically, it is from one of their powerful microscopes, the aptly-named “light sheet microscope”. And, so as not to keep you in suspense any longer, it is a mouse embryo, fixed for all eternity by the use of laser beams! But what for? For the purpose of studying innate lymphoid cells, a specific type of immune cell that combines the characteristics of T cells, adaptive immune cells, and innate immune cells.

  • Embryon de souris © CIML/Van de Pavert, Serge
    Mouse embryo viewed under a light sheet microscope. © CIML/Van de Pavert, Serge
  • Laser bleu © Inserm/Guénet, François
    What are the X-wings from Star Wars doing at the CIML? Nothing. These red and blue rays are sheets of laser light hitting each side of the mouse embryo being examined. Its lipids and water have been removed, rendering it transparent. Depending on the wavelength (and therefore the color) of the laser, the fluorescent-stained molecules or cells will react. They will become excited and visible, one after the other, in order to create these multicolored images. © Inserm/Guénet, François
  • Laser rouge © Inserm/Guénet, François
    What are the X-wings from Star Wars doing at the CIML? Nothing. These red and blue rays are sheets of laser light hitting each side of the mouse embryo being examined. Its lipids and water have been removed, rendering it transparent. Depending on the wavelength (and therefore the color) of the laser, the fluorescent-stained molecules or cells will react. They will become excited and visible, one after the other, in order to create these multicolored images. © Inserm/Guénet, François
  • Embroyon de souris © Inserm/Guénet, François
    In green, a mouse embryo, excited by a yellow-green laser: this is what we can see with the naked eye, looking down. © Inserm/Guénet, François
  • Chercheur © Inserm/Guénet, François
    Thanks to this embedding station, each tissue specimen is dehydrated before being coated in paraffin by Lionel Chasson, Technical Manager of the Histology platform at the CIML. An operation essential in order to preserve the specimen and apply the treatments that will make the cells or structural organ defects in question visible. © Inserm/Guénet, François
  • Bacs de coloration © Inserm/Guénet, François
    In the slide stainer, the sample is plunged into one color bath after another, each making it possible to reveal a different element: hematoxylin stains the cell nuclei violet, eosin stains the cytoplasm red, trichrome stains the collagen blue… © Inserm/Guénet, François
  • Technique d'immunomarquage © Inserm/Guénet, François
    This paraffin block was sliced using a microtome, at room temperature. Once the specimen is reached, each collected section is placed on a slide, to which a drop of water is added first. The slide is then heated on a special plate to 60°C in order to soften the paraffin. The slides are then stained before going under the microscope. © Inserm/Guénet, François
  • Paraffine © Inserm/Guénet, François
    Depending on the analysis required, in particular the immunolabeling techniques, it is necessary to coat the sample, here a mouse spleen, in a gel enabling it to be frozen without affecting cell or tissue integrity. The cryostat then enables the scientist to slice and then retrieve, using a brush, slices of the sample that are thin enough to allow light to pass through and be viewed under an optical or fluorescence microscope. © Inserm/Guénet, François
  • Chercheur © Inserm/Guénet, François
    A worm sorter - what for? Because the Caenorhabditis elegans nematode is the tiniest of the animal models used at the CIML: transparent and just one millimeter long, it is made up of a specific number of cells, which are easy to track if they are labeled with fluorescent molecules. How? Thanks to this machine operated by Jérôme Belougne, the Technical Manager of this platform. In the wells, stained red, worms are wriggling. The machine sorts them in accordance with the fluorescence that they do or do not express, which is proof, for example, that a certain version of a gene has been introduced by the researchers. © Inserm/Guénet, François
  • Chercheur © Inserm/Guénet, François
    On the screen Jérôme Belougne checks the presence of the nematodes in the wells, photographed by a programmed robot. A system which is used to automate the monitoring of worm growth and survival. © Inserm/Guénet, François
  • Chercheur © Inserm/Guénet, François
    Super-resolution microscopy makes it possible to break the 200-nm limit on image resolution. These days, it is possible to get around this by localizing the individual fluorescent molecules on a series of several tens of thousands of images in order to reconstitute a super-resolution image with the precision of several tens of nanometers. The dSTORM technique used at the CIML is based on the use of a redox medium in which the cells are imaged, together with powerful lasers, whose alignment is being checked by Sébastien Mailfert, Co-Technical Manager of the ImagImm photonic imaging platform. © Inserm/Guénet, François

Serge Van de Pavert, author of the photo, and his team are researching a subtype of these cells involved in the development of secondary lymphoid organs (such as the spleen and lymph nodes), sites of transit, accumulation, and meeting of immune cells and antigens recognized by antibodies. From the embryonic stage, their formation and differentiation can be modified by changing certain dietary factors. The purpose of this 3D imaging with immunofluorescent labeling is therefore to analyze the number and distribution of the cells involved in building the lymphatic system, which is essential for tissue drainage, the circulation of nutrients, hormones, and white blood cells, for immune system function and for healing.

From worm to man, from molecules to the whole organism, from physiology to pathology, the CIML and its 14 teams address all areas of contemporary immunology: the genesis of numerous cell populations, their modes of differentiation and activation, their involvement in cancer, infectious and inflammatory diseases, and the mechanisms of cell death.

Find the report in issue 39 of Science&Santé magazine