[{"day":"02","file":[{"file_name":"PhDThesis_BettinaZens_2023_final.pdf","creator":"bzens","content_type":"application/pdf","relation":"main_file","embargo":"2024-02-07","file_size":23082464,"checksum":"069d87f025e0799bf9e3c375664264f2","date_updated":"2024-02-08T23:30:04Z","file_id":"12527","access_level":"open_access","date_created":"2023-02-07T13:07:38Z"},{"date_created":"2023-02-07T13:09:05Z","access_level":"closed","date_updated":"2024-02-08T23:30:04Z","file_id":"12528","checksum":"8c66ed203495d6e078ed1002a866520c","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","relation":"source_file","file_size":106169509,"creator":"bzens","embargo_to":"open_access","file_name":"PhDThesis_BettinaZens_2023_final.docx"}],"author":[{"first_name":"Bettina","last_name":"Zens","full_name":"Zens, Bettina","id":"45FD126C-F248-11E8-B48F-1D18A9856A87"}],"degree_awarded":"PhD","title":"Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography","department":[{"_id":"GradSch"},{"_id":"FlSc"}],"user_id":"8b945eb4-e2f2-11eb-945a-df72226e66a9","publisher":"Institute of Science and Technology Austria","article_processing_charge":"No","publication_identifier":{"isbn":["978-3-99078-027-5"],"issn":["2663-337X"]},"doi":"10.15479/at:ista:12491","project":[{"_id":"eba3b5f6-77a9-11ec-83b8-cf0905748aa3","name":"Integrated visual proteomics of reciprocal cell-extracellular matrix interactions"},{"name":"NÖ-Fonds Preis für die Jungforscherin des Jahres am IST Austria","_id":"059B463C-7A3F-11EA-A408-12923DDC885E"}],"language":[{"iso":"eng"}],"keyword":["cryo-EM","cryo-ET","FIB milling","method development","FIBSEM","extracellular matrix","ECM","cell-derived matrices","CDMs","cell culture","high pressure freezing","HPF","structural biology","tomography","collagen"],"abstract":[{"text":"The extracellular matrix (ECM) is a hydrated and complex three-dimensional network consisting of proteins, polysaccharides, and water. It provides structural scaffolding for the cells embedded within it and is essential in regulating numerous physiological processes, including cell migration and proliferation, wound healing, and stem cell fate. \r\nDespite extensive study, detailed structural knowledge of ECM components in physiologically relevant conditions is still rudimentary. This is due to methodological limitations in specimen preparation protocols which are incompatible with keeping large samples, such as the ECM, in their native state for subsequent imaging. Conventional electron microscopy (EM) techniques rely on fixation, dehydration, contrasting, and sectioning. This results in the alteration of a highly hydrated environment and the potential introduction of artifacts. Other structural biology techniques, such as nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, allow high-resolution analysis of protein structures but only work on homogenous and purified samples, hence lacking contextual information. Currently, no approach exists for the ultrastructural and structural study of extracellular components under native conditions in a physiological, 3D environment. \r\nIn this thesis, I have developed a workflow that allows for the ultrastructural analysis of the ECM in near-native conditions at molecular resolution. The developments I introduced include implementing a novel specimen preparation workflow for cell-derived matrices (CDMs) to render them compatible with ion-beam milling and subsequent high-resolution cryo-electron tomography (ET). \r\nTo this end, I have established protocols to generate CDMs grown over several weeks on EM grids that are compatible with downstream cryo-EM sample preparation and imaging techniques. Characterization of these ECMs confirmed that they contain essential ECM components such as collagen I, collagen VI, and fibronectin I in high abundance and hence represent a bona fide biologically-relevant sample. I successfully optimized vitrification of these specimens by testing various vitrification techniques and cryoprotectants. \r\nIn order to obtain high-resolution molecular insights into the ultrastructure and organization of CDMs, I established cryo-focused ion beam scanning electron microscopy (FIBSEM) on these challenging and complex specimens. I explored different approaches for the creation of thin cryo-lamellae by FIB milling and succeeded in optimizing the cryo-lift-out technique, resulting in high-quality lamellae of approximately 200 nm thickness. \r\nHigh-resolution Cryo-ET of these lamellae revealed for the first time the architecture of native CDM in the context of matrix-secreting cells. This allowed for the in situ visualization of fibrillar matrix proteins such as collagen, laying the foundation for future structural and ultrastructural characterization of these proteins in their near-native environment. \r\nIn summary, in this thesis, I present a novel workflow that combines state-of-the-art cryo-EM specimen preparation and imaging technologies to permit characterization of the ECM, an important tissue component in higher organisms. This innovative and highly versatile workflow will enable addressing far-reaching questions on ECM architecture, composition, and reciprocal ECM-cell interactions.","lang":"eng"}],"date_updated":"2024-02-08T23:30:05Z","month":"02","type":"dissertation","oa_version":"Published Version","page":"187","date_created":"2023-02-02T14:50:20Z","file_date_updated":"2024-02-08T23:30:04Z","year":"2023","_id":"12491","has_accepted_license":"1","oa":1,"publication_status":"published","acknowledged_ssus":[{"_id":"EM-Fac"},{"_id":"LifeSc"},{"_id":"Bio"}],"supervisor":[{"last_name":"Schur","first_name":"Florian KM","orcid":"0000-0003-4790-8078","id":"48AD8942-F248-11E8-B48F-1D18A9856A87","full_name":"Schur, Florian KM"}],"ddc":["570"],"date_published":"2023-02-02T00:00:00Z","status":"public","alternative_title":["ISTA Thesis"],"citation":{"chicago":"Zens, Bettina. “Ultrastructural Characterization of Natively Preserved Extracellular Matrix by Cryo-Electron Tomography.” Institute of Science and Technology Austria, 2023. <a href=\"https://doi.org/10.15479/at:ista:12491\">https://doi.org/10.15479/at:ista:12491</a>.","ieee":"B. Zens, “Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography,” Institute of Science and Technology Austria, 2023.","short":"B. Zens, Ultrastructural Characterization of Natively Preserved Extracellular Matrix by Cryo-Electron Tomography, Institute of Science and Technology Austria, 2023.","ama":"Zens B. Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography. 2023. doi:<a href=\"https://doi.org/10.15479/at:ista:12491\">10.15479/at:ista:12491</a>","apa":"Zens, B. (2023). <i>Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography</i>. Institute of Science and Technology Austria. <a href=\"https://doi.org/10.15479/at:ista:12491\">https://doi.org/10.15479/at:ista:12491</a>","mla":"Zens, Bettina. <i>Ultrastructural Characterization of Natively Preserved Extracellular Matrix by Cryo-Electron Tomography</i>. Institute of Science and Technology Austria, 2023, doi:<a href=\"https://doi.org/10.15479/at:ista:12491\">10.15479/at:ista:12491</a>.","ista":"Zens B. 2023. Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography. Institute of Science and Technology Austria."},"related_material":{"record":[{"relation":"part_of_dissertation","id":"8586","status":"public"}]}},{"project":[{"grant_number":"P33367","name":"Structure and isoform diversity of the Arp2/3 complex","_id":"9B954C5C-BA93-11EA-9121-9846C619BF3A"},{"_id":"059B463C-7A3F-11EA-A408-12923DDC885E","name":"NÖ-Fonds Preis für die Jungforscherin des Jahres am IST Austria"}],"issue":"3","language":[{"iso":"eng"}],"keyword":["electron microscopy","cryo-EM","EM sample preparation","3D printing","cell culture"],"isi":1,"publication_identifier":{"issn":["1047-8477"]},"quality_controlled":"1","doi":"10.1016/j.jsb.2020.107633","department":[{"_id":"FlSc"}],"user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","publisher":"Elsevier","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","short":"CC BY (4.0)"},"article_type":"original","scopus_import":"1","article_processing_charge":"Yes (via OA deal)","publication":"Journal of Structural Biology","day":"01","file":[{"checksum":"c48cbf594e84fc2f91966ffaafc0918c","file_id":"8937","date_updated":"2020-12-10T14:01:10Z","access_level":"open_access","date_created":"2020-12-10T14:01:10Z","file_name":"2020_JourStrucBiology_Faessler.pdf","success":1,"creator":"dernst","file_size":7076870,"content_type":"application/pdf","relation":"main_file"}],"author":[{"id":"404F5528-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-7149-769X","full_name":"Fäßler, Florian","last_name":"Fäßler","first_name":"Florian"},{"full_name":"Zens, Bettina","id":"45FD126C-F248-11E8-B48F-1D18A9856A87","last_name":"Zens","first_name":"Bettina"},{"first_name":"Robert","last_name":"Hauschild","orcid":"0000-0001-9843-3522","id":"4E01D6B4-F248-11E8-B48F-1D18A9856A87","full_name":"Hauschild, Robert"},{"id":"48AD8942-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-4790-8078","full_name":"Schur, Florian KM","first_name":"Florian KM","last_name":"Schur"}],"article_number":"107633","title":"3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy","status":"public","external_id":{"isi":["000600997800008"]},"citation":{"short":"F. Fäßler, B. Zens, R. Hauschild, F.K. Schur, Journal of Structural Biology 212 (2020).","ieee":"F. Fäßler, B. Zens, R. Hauschild, and F. K. Schur, “3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy,” <i>Journal of Structural Biology</i>, vol. 212, no. 3. Elsevier, 2020.","chicago":"Fäßler, Florian, Bettina Zens, Robert Hauschild, and Florian KM Schur. “3D Printed Cell Culture Grid Holders for Improved Cellular Specimen Preparation in Cryo-Electron Microscopy.” <i>Journal of Structural Biology</i>. Elsevier, 2020. <a href=\"https://doi.org/10.1016/j.jsb.2020.107633\">https://doi.org/10.1016/j.jsb.2020.107633</a>.","mla":"Fäßler, Florian, et al. “3D Printed Cell Culture Grid Holders for Improved Cellular Specimen Preparation in Cryo-Electron Microscopy.” <i>Journal of Structural Biology</i>, vol. 212, no. 3, 107633, Elsevier, 2020, doi:<a href=\"https://doi.org/10.1016/j.jsb.2020.107633\">10.1016/j.jsb.2020.107633</a>.","ista":"Fäßler F, Zens B, Hauschild R, Schur FK. 2020. 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy. Journal of Structural Biology. 212(3), 107633.","apa":"Fäßler, F., Zens, B., Hauschild, R., &#38; Schur, F. K. (2020). 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy. <i>Journal of Structural Biology</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.jsb.2020.107633\">https://doi.org/10.1016/j.jsb.2020.107633</a>","ama":"Fäßler F, Zens B, Hauschild R, Schur FK. 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy. <i>Journal of Structural Biology</i>. 2020;212(3). doi:<a href=\"https://doi.org/10.1016/j.jsb.2020.107633\">10.1016/j.jsb.2020.107633</a>"},"related_material":{"record":[{"relation":"used_in_publication","status":"public","id":"14592"},{"id":"12491","status":"public","relation":"dissertation_contains"}]},"intvolume":"       212","has_accepted_license":"1","publication_status":"published","oa":1,"acknowledged_ssus":[{"_id":"ScienComp"},{"_id":"LifeSc"},{"_id":"Bio"},{"_id":"EM-Fac"}],"date_published":"2020-12-01T00:00:00Z","ddc":["570"],"year":"2020","acknowledgement":"This work was supported by the Austrian Science Fund (FWF, P33367) to FKMS. BZ acknowledges support by the Niederösterreich Fond. This research was also supported by the Scientific Service Units (SSU) of IST Austria through resources provided by Scientific Computing (SciComp), the Life Science Facility (LSF), the BioImaging Facility (BIF) and the Electron Microscopy Facility (EMF). We thank Georgi Dimchev (IST Austria) and Sonja Jacob (Vienna Biocenter Core Facilities) for testing our grid holders in different experimental setups and Daniel Gütl and the Kondrashov group (IST Austria) for granting us repeated access to their 3D printers. We also thank Jonna Alanko and the Sixt lab (IST Austria) for providing us HeLa cells, primary BL6 mouse tail fibroblasts, NIH 3T3 fibroblasts and human telomerase immortalised foreskin fibroblasts for our experiments. We are thankful to Ori Avinoam and William Wan for helpful comments on the manuscript and also thank Dorotea Fracchiolla (Art&Science) for illustrating the graphical abstract.","_id":"8586","oa_version":"Published Version","month":"12","type":"journal_article","abstract":[{"lang":"eng","text":"Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications."}],"date_updated":"2024-03-25T23:30:04Z","file_date_updated":"2020-12-10T14:01:10Z","date_created":"2020-09-29T13:24:06Z","volume":212}]