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Unravelling its structure and dynamics is therefore essential f we are to understand the mechanics of these processes and their effects in development and disease. Chromatin is, however, a difficult target to study: it is found in a crowded environment within the nucleus, is structurally organized on multiple length scales, is variable within and between nuclei, and is highly dynamic. Capturing this information requires instrumentation which can (i) measure on multiple length scales, from the whole cell down to tens of nm, (ii) follow chromatin dynamics in living cells, and (iii) acquire and quantify thousands of images in a manageable time frame to overcome the intrinsic variability and provide a statistical description of chromatin behavior. Such an instrument does not yet exist, with existing instrumentation being limited in resolution, dynamic speed, and throughput. We propose an innovative multi-disciplinary approach that combines developments in optics, data processing and modeling to realize an integrated system for automated high-throughput super- resolution imaging and dense single-molecule tracking in the cell nucleus. Our Specific Aims are: 1) Develop an automated multicolor 3D single-molecule switching (SMS) nanoscope for dynamic imaging and particle-tracking in the nucleus, 2) Develop data processing tools for high-throughput 3D- SMS nanoscopy of 100-1,000 cells/h, 3) Develop chromatin modeling tools that take advantage of the unprecedented level of detail and statistical depth of the 4D data provided by high-throughput particle- tracking and SMS nanoscopy, and 4) test the performance and refine our technical developments by applying them to a diverse set of representative and important questions in the field of chromatin architecture, including the mobility and dynamics of transcription factors and nucleosomes, and the processes of synaptonemal assembly and telomere recombination. The proposal represents a fundamental departure from the traditional view of the nanoscopy image generation procedure as a hands-on process heavily involving an expert user to an automated, high- throughput method with focus on quantification and efficiency. 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(2023)", "ID": "PMID:36914886", "date_published": "2023-03-13", "authors": ["Barentine AES", "Lin Y", "Courvan EM", "Kidd P", "Liu M", "Balduf L", "Phan T", "Rivera-Molina F", "Grace MR", "Marin Z", "Lessard M", "Rios Chen J", "Wang S", "Neugebauer KM", "Bewersdorf J", "Baddeley D"], "title": "An integrated platform for high-throughput nanoscopy.", "@type": ["Publication", "Item"], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "publications_of_exp": [{"uuid": "d698acc0-9564-4df9-b3a4-1b698ed03d84", "@id": "/publications/d698acc0-9564-4df9-b3a4-1b698ed03d84/", "ID": "PMID:36914886", "display_title": "Barentine AES et al. (2023) PMID:36914886", "journal": "Nature biotechnology", "@type": ["Publication", "Item"], "short_attribution": "Barentine AES et al. (2023)", "date_published": "2023-03-13", "title": "An integrated platform for high-throughput nanoscopy.", "abstract": "Single-molecule localization microscopy enables three-dimensional fluorescence  imaging at tens-of-nanometer resolution, but requires many camera frames to  reconstruct a super-resolved image. This limits the typical throughput to tens of  cells per day. While frame rates can now be increased by over an order of  magnitude, the large data volumes become limiting in existing workflows. Here we  present an integrated acquisition and analysis platform leveraging  microscopy-specific data compression, distributed storage and distributed  analysis to enable an acquisition and analysis throughput of 10,000 cells per  day. The platform facilitates graphically reconfigurable analyses to be  automatically initiated from the microscope during acquisition and remotely  executed, and can even feed back and queue new acquisition tasks on the  microscope. We demonstrate the utility of this framework by imaging hundreds of  cells per well in multi-well sample formats. Our platform, implemented within the  PYthon-Microscopy Environment (PYME), is easily configurable to control custom  microscopes, and includes a plugin framework for user-defined extensions.", "authors": ["Barentine AES", "Lin Y", "Courvan EM", "Kidd P", "Liu M", "Balduf L", "Phan T", "Rivera-Molina F", "Grace MR", "Marin Z", "Lessard M", "Rios Chen J", "Wang S", "Neugebauer KM", "Bewersdorf J", "Baddeley D"], "status": "current", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}], "experiment_categorizer": {"field": "Target", "value": "GRCh38:5:116144601-116449681 region, Chromosome 22 TADs", "combined": "Target: GRCh38:5:116144601-116449681 region, Chromosome 22 TADs"}, "experiment_summary": "DNA FISH on IMR-90 (Tier 1)", "@context": "/terms/", "aggregated-items": {"badges": [{"parent": "/biosamples/4DNBSB4KJ2BK/", "embedded_path": "biosample.badges", "item": {"messages": ["Biosample receives gold status for being a 4DN Tier 1 or Tier 2 cell line that follows the approved SOP and contains all of the pertinent metadata information as required by the 4DN Samples working group."], "badge": {"commendation": "Gold Biosample", "warning": null, "uuid": "6c2b7409-4478-4e15-aabc-1a0df6b883e9", "@id": "/badges/gold-biosample/", "badge_icon": "/static/img/badges/biosample-badge-gold-star.svg", "description": "Gold biosample"}}}]}, "validation-errors": []}