---
_id: '12491'
abstract:
- lang: eng
  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."
acknowledged_ssus:
- _id: EM-Fac
- _id: LifeSc
- _id: Bio
alternative_title:
- ISTA Thesis
article_processing_charge: No
author:
- first_name: Bettina
  full_name: Zens, Bettina
  id: 45FD126C-F248-11E8-B48F-1D18A9856A87
  last_name: Zens
citation:
  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>
  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.
  ista: Zens B. 2023. Ultrastructural characterization of natively preserved extracellular
    matrix by cryo-electron tomography. Institute of Science and Technology Austria.
  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>.
  short: B. Zens, Ultrastructural Characterization of Natively Preserved Extracellular
    Matrix by Cryo-Electron Tomography, Institute of Science and Technology Austria,
    2023.
date_created: 2023-02-02T14:50:20Z
date_published: 2023-02-02T00:00:00Z
date_updated: 2024-02-08T23:30:05Z
day: '02'
ddc:
- '570'
degree_awarded: PhD
department:
- _id: GradSch
- _id: FlSc
doi: 10.15479/at:ista:12491
file:
- access_level: open_access
  checksum: 069d87f025e0799bf9e3c375664264f2
  content_type: application/pdf
  creator: bzens
  date_created: 2023-02-07T13:07:38Z
  date_updated: 2024-02-08T23:30:04Z
  embargo: 2024-02-07
  file_id: '12527'
  file_name: PhDThesis_BettinaZens_2023_final.pdf
  file_size: 23082464
  relation: main_file
- access_level: closed
  checksum: 8c66ed203495d6e078ed1002a866520c
  content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document
  creator: bzens
  date_created: 2023-02-07T13:09:05Z
  date_updated: 2024-02-08T23:30:04Z
  embargo_to: open_access
  file_id: '12528'
  file_name: PhDThesis_BettinaZens_2023_final.docx
  file_size: 106169509
  relation: source_file
file_date_updated: 2024-02-08T23:30:04Z
has_accepted_license: '1'
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
language:
- iso: eng
month: '02'
oa: 1
oa_version: Published Version
page: '187'
project:
- _id: eba3b5f6-77a9-11ec-83b8-cf0905748aa3
  name: Integrated visual proteomics of reciprocal cell-extracellular matrix interactions
- _id: 059B463C-7A3F-11EA-A408-12923DDC885E
  name: NÖ-Fonds Preis für die Jungforscherin des Jahres am IST Austria
publication_identifier:
  isbn:
  - 978-3-99078-027-5
  issn:
  - 2663-337X
publication_status: published
publisher: Institute of Science and Technology Austria
related_material:
  record:
  - id: '8586'
    relation: part_of_dissertation
    status: public
status: public
supervisor:
- first_name: Florian KM
  full_name: Schur, Florian KM
  id: 48AD8942-F248-11E8-B48F-1D18A9856A87
  last_name: Schur
  orcid: 0000-0003-4790-8078
title: Ultrastructural characterization of natively preserved extracellular matrix
  by cryo-electron tomography
type: dissertation
user_id: 8b945eb4-e2f2-11eb-945a-df72226e66a9
year: '2023'
...