Confocal Raman-in-SEM Imaging: a New Method for 3D Morphology of Asbestos- like Fibers in a Mineral Matrix -Complementarity with SEM-FIB

International audienceAsbestos consists of natural mineral fibers crystallized in a specific way with specific properties including flexibility, high tensile strength, resistance to heat and chemical degradation. The term asbestos refers to one fibrous serpentine, and five asbestiform varieties of amphiboles [1][2]. Asbestos is considered a Category 1 human carcinogen (e.g. [1][3][4][5][6]), as inhalation of asbestos fibers causes respiratory diseases, in particular asbestosis, lung cancers and malignant mesothelioma. Due to the pathogenicity of asbestos fibers, their use has been banned in most countries around the world. The classification as "asbestos" comprises three main characteristics: sub-micrometric fibrous morphology (so-called asbestiform), chemistry and crystallography (i.e. mineralogy). The ban applies to natural materials, which are quarried and used for public works projects. Compliance with existing rules requires accurate diagnosis of the asbestos or non-asbestos nature of the materials likely to be exploited in a quarry or involved in the opening of a construction site. Conventional methods for asbestos diagnosis, mainly based on the use of TEM, are hard to apply to massive materials as the sample preparation protocol is complex, it is likely to release artifacts like cleavage fragments, or sample-piece thinning is costly and hard to achieve and thus not applicable in routine analysis. The use of a LV-FE-SEM-EDS (Low vacuum field emission gun scannning electron microscope equiped with energy dispersive spectroscopy) coupled to confocal Raman-in-SEM imaging (RISE) is a new and efficient method for identifying the mineral nature of sub-micrometer fibers or fibrous bundles. SEM coupled to EDS provides a qualitative or semi-quantitative composition of the fiber. The crystal structure is determined by confocal Raman spectroscopy (with lateral resolution down to 360 nm [7]). Thus the combination of these two kinds of information allows for accurate identification of both the nature and the morphology of the mineral. The combination of SEM, EDS and confocal Raman imaging in a unique analytical system (RISE) allows precise location of the same area / fiber and identification of its mineral nature. In addition, RISE allows the acquisition of 3D data, which is able to provide morphological information of the mineral distribution in the sample volume and leading to the determination of the aspect ratio, a critical parameter for asbestos (i.e. asbestiform or non-asbestiform). Moreover, this combination of techniques is not destructive. The coupling of SEM-EDS with RISE is a powerful analytical system that simplifies and reinforces existing analytical procedures. For highest resolution studies of fibers in the sample volume (3 D imaging), the RISE can be connected to a FIB-SEM [7]. Although FIB volume reconstruction is a destructive method, the resolution of the voxel can be extended to below 100 nm [8] – a resolution sufficient for visualizing even the thinnest fibers. Furthermore, Raman 3D view is an efficient tool for sampling TEM slices using the FIB

Thermus and the Pink Discoloration Defect in Cheese

peer-reviewedA DNA sequencing-based strategy was applied to study the microbiology of Continental-type cheeses with a pink discoloration defect. The basis for this phenomenon has remained elusive, despite decades of research. The bacterial composition of cheese containing the defect was compared to that of control cheese using 16S rRNA gene and shotgun metagenomic sequencing as well as quantitative PCR (qPCR). Throughout, it was apparent that Thermus, a carotenoid-producing genus, was present at higher levels in defect-associated cheeses than in control cheeses. Prompted by this finding and data confirming the pink discoloration to be associated with the presence of a carotenoid, a culture-based approach was employed, and Thermus thermophilus was successfully cultured from defect-containing cheeses. The link between Thermus and the pinking phenomenon was then established through the cheese defect equivalent of Koch’s postulates when the defect was recreated by the reintroduction of a T. thermophilus isolate to a test cheese during the manufacturing process. IMPORTANCE Pink discoloration in cheese is a defect affecting many cheeses throughout the world, leading to significant financial loss for the dairy industry. Despite decades of research, the cause of this defect has remained elusive. The advent of high-throughput, next-generation sequencing has revolutionized the field of food microbiology and, with respect to this study, provided a means of testing a possible microbial basis for this defect. In this study, a combined 16S rRNA, whole-genome sequencing, and quantitative PCR approach was taken. This resulted in the identification of Thermus, a carotenoid-producing thermophile, in defect-associated cheeses and the recreation of the problem in cheeses to which Thermus was added. This finding has the potential to lead to new strategies to eliminate this defect, and our method represents an approach that can be employed to investigate the role of microbes in other food defects of unknown origin.Teagasc Walsh Fellowship Programm