Comparative molecular dark field modes in cell imaging

International audienceA way to minimize chemical heavy contrastant agent and observing cells as close as possible from native conditions, even using classic chemical fixative procedure, is to increase the imaging contrast using diffraction contrast imaging. We have investigated the imaging contrast study of cell ultrastructure components at the molecular level using conventional and unconventional imaging methods

Dark Field Transmission Electron Microscopy Imaging for Biological and Soft Matter Systems

International audienceIn this study, we present a new application of the transmission electron microscopy dark field mode for cell imaging. We have applied this imaging mode to two types of cellular systems: human HeLa cells to analyze molecular membrane systems and HC11 mouse mammary cells containing lipid molecule droplets. We have also studied a third macromolecular system, copolymer nanoparticles for the characterization of core-shell structures. We want to show the effective use of diffraction contrast, even on amorphous systems for increasing the image contrast and the signal/noise ratio. We discuss the TEM dark field advantages for the analysis of polymers and other macromolecular systems, including biological, systems compared to the bright field mode

Une étude de la phénologie de la végétation dominante dans une tourbière à sphaignes de montagne avec Sentinel-2 et des observations

International audienceAbstract Peatlands store more than a third of the global soil organic carbon stock. Bryophytes, more specifically Sphagnum mosses, play a major role in the carbon and water cycles of these ecosystems. There is a need to include Sphagnum mosses into Earth system models to better simulate the dynamics of peatlands in a changing environment. Leaf area index (LAI) is a key plant trait that characterizes the plant photosynthesizing capacity. Moreover, LAI is a variable calculated by land surface models used in climate models, allowing control of the exchange of matter and energy between vegetation and the environment. There is extremely little data on Sphagnum LAI and none on its seasonal change. We monitored Sphagnum mosses LAI phenology in a mountainous peatland site (altitude of 1,343 m) from June to December 2021 using two methods: 2D scans of monthly Sphagnum moss samples and analysis of Sentinel‐2 images. LAI derived from field campaigns and the remote sensing approach show a strong seasonality, with high peak values reaching 10 and 7 m 2 ·m −2 , respectively. The Sentinel‐2 images were also used to derive common vegetation indices. The moisture soil index effectively discriminates Sphagnum ‐dominated areas in the peatland. Satellite‐derived LAI of Sphagnum mosses is directly correlated to gross primary production monitored by gas exchange measurements ( R 2 = 0.83) but also to physical drivers of the environment such as air temperature ( R 2 = 0.74) or water table depth ( R 2 = 0.61) over the 2017–2021 period. It is therefore highly suitable to investigate ecosystemic functions

Comparaison des modes d'imagerie fond noir TEM et STEM pour l'observation de structures moléculaires

International audienceUne façon de minimiser les agents lourds des contrastants chimiques et d’observer les cellules proche de leur état natif, même en utilisant la procédure classique du fixateur chimique, est d'augmenter le contraste de diffraction ou le contraste de diffusion pour l’imagerie. A titre d'exemple, dans le domaine particulier des polymères, le mode fond noir haute résolution (DF-HRTEM) a permis d'observer les unités structurales de monomères [1]. De plus, comme le montre l’étude quantitative STEM [2] sur des échantillons biologiques, les contrastes STEM sont corrélés à la chimie des phases ce qui permet d'identifier la présence et l'emplacement des composants moléculaires dans une cellule, comme l'ADN, protéines, lipides, complexes nucléoprotéiques etc ... Nous voulons étudier les composants de la structure des cellules au niveau moléculaire en utilisant des modes conventionnels et non conventionnels. Nous présentons les résultats d'une étude comparative TEM et STEM sur cellulehumaine Hela, en utilisant les modes d'imagerie STEM fond noir (ADF, HAADF) et TEM fond noir haute résolution (DF-MEHR). Les modes d'imagerie STEM donnent des informations de la structure, à partir de la diffusion des électrons (ADF), et de la chimie locale à l'échelle atomique ou moléculaire (HAADF). Le mode fond noir DF-MEHR en TEM donne des informations structurales combinant le contraste de phase et le contraste d'amplitude comparable aux conditions d'imagerie haute résolution HRTEM, mais en utilisant le contraste minimum de défocalisation de l’objectif. Nous voulons aussi tester la limite de résolution des informations pour l'analyse d'imagerie cellulaire, en utilisant la nouvelle génération de microscopes TEM / STEM équipé de canon à émission de champ et de correcteurs d'aberration. Différents microscopes TEM et STEM ont été utilisés pour cette étude. Le mode d'imagerie en fond noir haute résolution (DF-HRTEM) a été réalisé sur un Zeiss 912 (120KeV), Hitachi HU12A (75KeV), et un CM20 FEG Twin (120KeV). Les modes d’imagerie STEM HAADF et ADF ont été réalisés avec deux types de TEM / STEM: Tecnai F20(200 keV) équipé d'un FEG Schottky et un correcteur de Cs Image (TEM résolution 0.12nm), un microscope Jeol2200FS équipé d'un FEG Schottky et un Correcteur Cs de la sonde Céos (0.1nm résolution STEM) et enfin avec l'aide d'un microscope Hitachi SEM en mode STEM HAADF en basse tension (30 keV). Les observations ont été réalisées sur des cellules humaines HeLa enrobées dans l'épon, contrastées et non contrastées, (acétate d'uranyle 2%), dans le bloc de préparation des échantillons ou déposé sur le support de grille de carbone, avec ou sans citrate de plomb). Les résultats de ces modes d'imagerie en fond noir seront présentés en soulignant leur contribution spécifique dans le contenu de l'imagerie moléculaire des cellules (contraste de l'image, résolution de l'information) et comparés au mode classique avec contrastant chimique.References[1] A. Oberlin, J. Ayache and M. Guigon, Journal of Polymer Science. vol. 20, p.579 (1982).[2] E. Kellenberger, E. carlemalm, W. Villigier, M. Wurtz, C. Mory and C. Colliex. In Annals NewYork academy of Sciences. 483, 202-228 (1986).[3] This research was supported by RCCM the national network belonging to MRCT of CNRS and also by METSAthe national microscopy CNRS network

Phospholipase A2 Receptor 1 Epitope Spreading at Baseline Predicts Reduced Likelihood of Remission of Membranous Nephropathy

G.L. and P.R. contributed equally to this work.International audienceThe phospholipase A2 receptor (PLA2R1) is the major autoantigen in primary membranous nephropathy. Several PLA2R1 epitopes have been characterized, and a retrospective study identified PLA2R1 epitope spreading as a potential indicator of poor prognosis. Here, we analyzed the predictive value of anti-PLA2R1 antibody (PLA2R1-Ab) titers and epitope spreading in a prospective cohort of 58 patients positive for PLA2R1-Ab randomly allocated to rituximab (n=29) or antiproteinuric therapy alone (n=29). At baseline, the epitope profile (CysR, CysRC1, CysRC7, or CysRC1C7) did not correlate with age, sex, time from diagnosis, proteinuria, or serum albumin, but epitope spreading strongly correlated with PLA2R1-Ab titer (P<0.001). Ten (58.8%) of the 17 patients who had epitope spreading at baseline and were treated with rituximab showed reversal of epitope spreading at month 6. In adjusted analysis, epitope spreading at baseline was associated with a decreased remission rate at month 6 (odds ratio, 0.16; 95% confidence interval, 0.04 to 0.72; P=0.02) and last follow-up (median, 23 months; odds ratio, 0.14; 95% confidence interval, 0.03 to 0.64; P=0.01), independently from age, sex, baseline PLA2R1-Ab level, and treatment group. We propose that epitope spreading at baseline be considered in the decision for early therapeutic intervention in patients with primary membranous nephropathy

Stocks et flux de carbone de la tourbière de Bernadouze

National audienceCette présentation synthétise les travaux de mesure des stocks et des flux de carbone de la tourbière de Bernadouze dans le cadre de l'Observatoire Hommes-Milieux (OHM) Pyrénées Haut Vicdessos et du Service National d'Observation Tourbières (SNO Tourbières)

Stocks et flux de carbone de la tourbière de Bernadouze

National audienceCette présentation synthétise les travaux de mesure des stocks et des flux de carbone de la tourbière de Bernadouze dans le cadre de l'Observatoire Hommes-Milieux (OHM) Pyrénées Haut Vicdessos et du Service National d'Observation Tourbières (SNO Tourbières)

Novel ELISA for thrombospondin type 1 domain-containing 7A autoantibodies in membranous nephropathy

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Exposure to dietary lipid leads to rapid production of cytosolic lipid droplets near the brush border membrane

Intestinal absorption of dietary lipids involves their hydrolysis in the lumen of proximal intestine as well as uptake, intracellular transport and re-assembly of hydrolyzed lipids in enterocytes, leading to the formation and secretion of the lipoproteins chylomicrons and HDL. In this study, we examined the potential involvement of cytosolic lipid droplets (CLD) whose function in the process of lipid absorption is poorly understood. Intestinal lipid absorption was studied in mouse after gavage. Three populations of CLD were purified by density ultracentrifugations, as well as the brush border membranes, which were analyzed by western-blots. Immunofluorescent localization of membranes transporters or metabolic enzymes, as well as kinetics of CLD production, were also studied in intestine or Caco-2 cells. We isolated three populations of CLD (ranging from 15 to 1000 nm) which showed differential expression of the major lipid transporters scavenger receptor BI (SR-BI), cluster of differentiation 36 (CD-36), Niemann Pick C-like 1 (NPC1L1), and the ATP-binding cassette transporters ABCG5/G8 but also caveolin 2 and fatty acid binding proteins. The enzyme monoacylglycerol acyltransferase 2 (MGAT2) was identified in the brush border membrane (BBM) in addition to the endoplasmic reticulum, suggesting local synthesis of triglycerides and CLD at both places. We show a very fast production of CLD by enterocytes associated with a transfer of apical constituents as lipid transporters. Our findings suggest that following their uptake by enterocytes, lipids can be partially metabolized at the BBM and packaged into CLD for their transportation to the ER