Microscopy & Enteroids

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Project Summary - Core B - Microscopy and Enteroids (Prasad and Estes) The role of the Microscopy and Enteroid Core is to provide a centralized facility to serve the electron microscopy (EM), fluorescence microscopy, and enteroid culture requirements of all of the investigators in the Program Project. This integrated Core is cost-effective and facilitates orderly and efficient use of resources to complete the proposed projects in a timely fashion. In addition, the use of the core allows for consistency and quality control across all projects. The specific functions of the Core, as required routinely by the three projects, will include: (1) Specimen preparation for EM and fluorescent microscopy, including epifluorescence, confocal, and deconvolution microscopy; (2) EM imaging of fecal samples from norovirus-infected persons, virus-like recombinant particles (VLPs), VLP-carbohydrate, VLP-antibody complexes, virus produced from infected cultures, full-length p41 oligomers, and empty and p41-reconstituted nanodiscs; (3) EM imaging of stained and fixed thin sections of virus-infected cells to assess cell binding and replication; (4) Evaluation of purity, particle integrity, and concentration and suitability of samples for cryo-EM imaging and computer reconstructions; (5) Immune electron microscopy using gold labels; (6) Data acquisition, interpretation, sharing, and archiving from the EM, fluorescent, confocal and deconvolution microscope imaging; (7) Scheduling and day-to-day EM maintenance of EM, epifluorescence, confocal and deconvolution microscopes; (8) Cultivation and providing media and well-characterized human intestinal enteroid (HIE) cultures to all Projects; (9) Preparation and characterization of genetically modified enteroid lines for all Projects; and (10) Preparation and characterization of biosensor-expressing HIE lines to monitor infection or host cell processes. Core key personnel will interface with program project group leaders and other investigators to meet the microscopy and enteroid requirements of all Projects. Overall, the microscopy and enteroid core is a key component of success of the program project and will provide critical resources, enhance communication and comparability of results between different projects.