---
_id: '15146'
abstract:
- lang: eng
text: The extracellular matrix (ECM) serves as a scaffold for cells and plays an
essential role in regulating numerous cellular processes, including cell migration
and proliferation. Due to limitations in specimen preparation for conventional
room-temperature electron microscopy, we lack structural knowledge on how ECM
components are secreted, remodeled, and interact with surrounding cells. We have
developed a 3D-ECM platform compatible with sample thinning by cryo-focused ion
beam milling, the lift-out extraction procedure, and cryo-electron tomography.
Our workflow implements cell-derived matrices (CDMs) grown on EM grids, resulting
in a versatile tool closely mimicking ECM environments. This allows us to visualize
ECM for the first time in its hydrated, native context. Our data reveal an intricate
network of extracellular fibers, their positioning relative to matrix-secreting
cells, and previously unresolved structural entities. Our workflow and results
add to the structural atlas of the ECM, providing novel insights into its secretion
and assembly.
acknowledged_ssus:
- _id: LifeSc
- _id: ScienComp
- _id: EM-Fac
- _id: M-Shop
acknowledgement: "Open Access funding provided by IST Austria. We thank Armel Nicolas
and his team at the ISTA proteomics facility, Alois Schloegl, Stefano Elefante,
and colleagues at the ISTA Scientific Computing facility, Tommaso Constanzo and
Ludek Lovicar at the Electron Microsocpy Facility (EMF), and Thomas Menner at the
Miba Machine shop for their support. We also thank Wanda Kukulski (University of
Bern) as well as Darío Porley, Andreas Thader, and other members of the Schur group
for helpful discussions. Matt Swulius and Jessica Heebner provided great support
in using Dragonfly. We thank Dorotea Fracciolla (Art & Science) for support in figure
illustration.\r\n\r\nThis research was supported by the Scientific Service Units
of ISTA through resources provided by Scientific Computing, the Lab Support Facility,
and the Electron Microscopy Facility. We acknowledge funding support from the following
sources: Austrian Science Fund (FWF) grant P33367 (to F.K.M. Schur), the Federation
of European Biochemical Societies (to F.K.M. Schur), Niederösterreich (NÖ) Fonds
(to B. Zens), FWF grant E435 (to J.M. Hansen), European Research Council under the
European Union’s Horizon 2020 research (grant agreement No. 724373) (to M. Sixt),
and Jenny and Antti Wihuri Foundation (to J. Alanko). This publication has been
made possible in part by CZI grant DAF2021-234754 and grant DOI https://doi.org/10.37921/812628ebpcwg
from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community
Foundation (to F.K.M. Schur)."
article_number: e202309125
article_processing_charge: Yes (via OA deal)
article_type: original
author:
- first_name: Bettina
full_name: Zens, Bettina
id: 45FD126C-F248-11E8-B48F-1D18A9856A87
last_name: Zens
orcid: 0000-0002-9561-1239
- first_name: Florian
full_name: Fäßler, Florian
id: 404F5528-F248-11E8-B48F-1D18A9856A87
last_name: Fäßler
orcid: 0000-0001-7149-769X
- first_name: Jesse
full_name: Hansen, Jesse
id: 1063c618-6f9b-11ec-9123-f912fccded63
last_name: Hansen
orcid: 0000-0001-7967-2085
- first_name: Robert
full_name: Hauschild, Robert
id: 4E01D6B4-F248-11E8-B48F-1D18A9856A87
last_name: Hauschild
orcid: 0000-0001-9843-3522
- first_name: Julia
full_name: Datler, Julia
id: 3B12E2E6-F248-11E8-B48F-1D18A9856A87
last_name: Datler
orcid: 0000-0002-3616-8580
- first_name: Victor-Valentin
full_name: Hodirnau, Victor-Valentin
id: 3661B498-F248-11E8-B48F-1D18A9856A87
last_name: Hodirnau
orcid: 0000-0003-3904-947X
- first_name: Vanessa
full_name: Zheden, Vanessa
id: 39C5A68A-F248-11E8-B48F-1D18A9856A87
last_name: Zheden
orcid: 0000-0002-9438-4783
- first_name: Jonna H
full_name: Alanko, Jonna H
id: 2CC12E8C-F248-11E8-B48F-1D18A9856A87
last_name: Alanko
orcid: 0000-0002-7698-3061
- first_name: Michael K
full_name: Sixt, Michael K
id: 41E9FBEA-F248-11E8-B48F-1D18A9856A87
last_name: Sixt
orcid: 0000-0002-6620-9179
- first_name: Florian KM
full_name: Schur, Florian KM
id: 48AD8942-F248-11E8-B48F-1D18A9856A87
last_name: Schur
orcid: 0000-0003-4790-8078
citation:
ama: Zens B, Fäßler F, Hansen J, et al. Lift-out cryo-FIBSEM and cryo-ET reveal
the ultrastructural landscape of extracellular matrix. Journal of Cell Biology.
2024;223(6). doi:10.1083/jcb.202309125
apa: Zens, B., Fäßler, F., Hansen, J., Hauschild, R., Datler, J., Hodirnau, V.-V.,
… Schur, F. K. (2024). Lift-out cryo-FIBSEM and cryo-ET reveal the ultrastructural
landscape of extracellular matrix. Journal of Cell Biology. Rockefeller
University Press. https://doi.org/10.1083/jcb.202309125
chicago: Zens, Bettina, Florian Fäßler, Jesse Hansen, Robert Hauschild, Julia Datler,
Victor-Valentin Hodirnau, Vanessa Zheden, Jonna H Alanko, Michael K Sixt, and
Florian KM Schur. “Lift-out Cryo-FIBSEM and Cryo-ET Reveal the Ultrastructural
Landscape of Extracellular Matrix.” Journal of Cell Biology. Rockefeller
University Press, 2024. https://doi.org/10.1083/jcb.202309125.
ieee: B. Zens et al., “Lift-out cryo-FIBSEM and cryo-ET reveal the ultrastructural
landscape of extracellular matrix,” Journal of Cell Biology, vol. 223,
no. 6. Rockefeller University Press, 2024.
ista: Zens B, Fäßler F, Hansen J, Hauschild R, Datler J, Hodirnau V-V, Zheden V,
Alanko JH, Sixt MK, Schur FK. 2024. Lift-out cryo-FIBSEM and cryo-ET reveal the
ultrastructural landscape of extracellular matrix. Journal of Cell Biology. 223(6),
e202309125.
mla: Zens, Bettina, et al. “Lift-out Cryo-FIBSEM and Cryo-ET Reveal the Ultrastructural
Landscape of Extracellular Matrix.” Journal of Cell Biology, vol. 223,
no. 6, e202309125, Rockefeller University Press, 2024, doi:10.1083/jcb.202309125.
short: B. Zens, F. Fäßler, J. Hansen, R. Hauschild, J. Datler, V.-V. Hodirnau, V.
Zheden, J.H. Alanko, M.K. Sixt, F.K. Schur, Journal of Cell Biology 223 (2024).
corr_author: '1'
date_created: 2024-03-21T06:45:51Z
date_published: 2024-03-20T00:00:00Z
date_updated: 2025-09-04T13:17:16Z
day: '20'
ddc:
- '570'
department:
- _id: FlSc
- _id: MiSi
- _id: Bio
- _id: EM-Fac
doi: 10.1083/jcb.202309125
ec_funded: 1
external_id:
isi:
- '001264190100001'
pmid:
- '38506714'
file:
- access_level: open_access
checksum: 90d1984a93660735e506c2a304bc3f73
content_type: application/pdf
creator: dernst
date_created: 2024-03-25T12:52:04Z
date_updated: 2024-03-25T12:52:04Z
file_id: '15188'
file_name: 2024_JCB_Zens.pdf
file_size: 11907016
relation: main_file
success: 1
file_date_updated: 2024-03-25T12:52:04Z
has_accepted_license: '1'
intvolume: ' 223'
isi: 1
issue: '6'
language:
- iso: eng
month: '03'
oa: 1
oa_version: Published Version
pmid: 1
project:
- _id: 9B954C5C-BA93-11EA-9121-9846C619BF3A
grant_number: P33367
name: Structure and isoform diversity of the Arp2/3 complex
- _id: 7bd318a1-9f16-11ee-852c-cc9217763180
grant_number: E435
name: In Situ Actin Structures via Hybrid Cryo-electron Microscopy
- _id: 25FE9508-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '724373'
name: Cellular Navigation Along Spatial Gradients
- _id: 059B463C-7A3F-11EA-A408-12923DDC885E
name: "NÃ\x96-Fonds Preis für die Jungforscherin des Jahres am IST Austria"
- _id: 2615199A-B435-11E9-9278-68D0E5697425
grant_number: '21317'
name: Spatiotemporal regulation of chemokine-induced signalling in leukocyte chemotaxis
- _id: 62909c6f-2b32-11ec-9570-e1476aab5308
grant_number: CZI01
name: CryoMinflux-guided in-situ visual proteomics and structure determination
publication: Journal of Cell Biology
publication_identifier:
eissn:
- 1540-8140
issn:
- 0021-9525
publication_status: published
publisher: Rockefeller University Press
quality_controlled: '1'
scopus_import: '1'
status: public
title: Lift-out cryo-FIBSEM and cryo-ET reveal the ultrastructural landscape of extracellular
matrix
tmp:
image: /images/cc_by.png
legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
short: CC BY (4.0)
type: journal_article
user_id: 317138e5-6ab7-11ef-aa6d-ffef3953e345
volume: 223
year: '2024'
...
---
OA_place: publisher
_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
orcid: 0000-0002-9561-1239
citation:
ama: Zens B. Ultrastructural characterization of natively preserved extracellular
matrix by cryo-electron tomography. 2023. doi:10.15479/at:ista:12491
apa: Zens, B. (2023). Ultrastructural characterization of natively preserved
extracellular matrix by cryo-electron tomography. Institute of Science and
Technology Austria. https://doi.org/10.15479/at:ista:12491
chicago: Zens, Bettina. “Ultrastructural Characterization of Natively Preserved
Extracellular Matrix by Cryo-Electron Tomography.” Institute of Science and Technology
Austria, 2023. https://doi.org/10.15479/at:ista:12491.
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. Ultrastructural Characterization of Natively Preserved Extracellular
Matrix by Cryo-Electron Tomography. Institute of Science and Technology Austria,
2023, doi:10.15479/at:ista:12491.
short: B. Zens, Ultrastructural Characterization of Natively Preserved Extracellular
Matrix by Cryo-Electron Tomography, Institute of Science and Technology Austria,
2023.
corr_author: '1'
date_created: 2023-02-02T14:50:20Z
date_published: 2023-02-02T00:00:00Z
date_updated: 2026-04-07T13:49:23Z
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: "NÃ\x96-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: ba8df636-2132-11f1-aed0-ed93e2281fdd
year: '2023'
...
---
_id: '8586'
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.
acknowledged_ssus:
- _id: ScienComp
- _id: LifeSc
- _id: Bio
- _id: EM-Fac
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.
article_number: '107633'
article_processing_charge: Yes (via OA deal)
article_type: original
author:
- first_name: Florian
full_name: Fäßler, Florian
id: 404F5528-F248-11E8-B48F-1D18A9856A87
last_name: Fäßler
orcid: 0000-0001-7149-769X
- first_name: Bettina
full_name: Zens, Bettina
id: 45FD126C-F248-11E8-B48F-1D18A9856A87
last_name: Zens
orcid: 0000-0002-9561-1239
- first_name: Robert
full_name: Hauschild, Robert
id: 4E01D6B4-F248-11E8-B48F-1D18A9856A87
last_name: Hauschild
orcid: 0000-0001-9843-3522
- first_name: Florian KM
full_name: Schur, Florian KM
id: 48AD8942-F248-11E8-B48F-1D18A9856A87
last_name: Schur
orcid: 0000-0003-4790-8078
citation:
ama: Fäßler F, Zens B, Hauschild R, Schur FK. 3D printed cell culture grid holders
for improved cellular specimen preparation in cryo-electron microscopy. Journal
of Structural Biology. 2020;212(3). doi:10.1016/j.jsb.2020.107633
apa: Fäßler, F., Zens, B., Hauschild, R., & Schur, F. K. (2020). 3D printed
cell culture grid holders for improved cellular specimen preparation in cryo-electron
microscopy. Journal of Structural Biology. Elsevier. https://doi.org/10.1016/j.jsb.2020.107633
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.” Journal of Structural Biology. Elsevier,
2020. https://doi.org/10.1016/j.jsb.2020.107633.
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,”
Journal of Structural Biology, vol. 212, no. 3. Elsevier, 2020.
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.
mla: Fäßler, Florian, et al. “3D Printed Cell Culture Grid Holders for Improved
Cellular Specimen Preparation in Cryo-Electron Microscopy.” Journal of Structural
Biology, vol. 212, no. 3, 107633, Elsevier, 2020, doi:10.1016/j.jsb.2020.107633.
short: F. Fäßler, B. Zens, R. Hauschild, F.K. Schur, Journal of Structural Biology
212 (2020).
corr_author: '1'
date_created: 2020-09-29T13:24:06Z
date_published: 2020-12-01T00:00:00Z
date_updated: 2026-06-06T22:30:10Z
day: '01'
ddc:
- '570'
department:
- _id: FlSc
doi: 10.1016/j.jsb.2020.107633
external_id:
isi:
- '000600997800008'
pmid:
- '32987119'
file:
- access_level: open_access
checksum: c48cbf594e84fc2f91966ffaafc0918c
content_type: application/pdf
creator: dernst
date_created: 2020-12-10T14:01:10Z
date_updated: 2020-12-10T14:01:10Z
file_id: '8937'
file_name: 2020_JourStrucBiology_Faessler.pdf
file_size: 7076870
relation: main_file
success: 1
file_date_updated: 2020-12-10T14:01:10Z
has_accepted_license: '1'
intvolume: ' 212'
isi: 1
issue: '3'
keyword:
- electron microscopy
- cryo-EM
- EM sample preparation
- 3D printing
- cell culture
language:
- iso: eng
month: '12'
oa: 1
oa_version: Published Version
pmid: 1
project:
- _id: 9B954C5C-BA93-11EA-9121-9846C619BF3A
grant_number: P33367
name: Structure and isoform diversity of the Arp2/3 complex
- _id: 059B463C-7A3F-11EA-A408-12923DDC885E
name: NÖ-Fonds Preis für die Jungforscherin des Jahres am IST Austria
publication: Journal of Structural Biology
publication_identifier:
issn:
- 1047-8477
publication_status: published
publisher: Elsevier
quality_controlled: '1'
related_material:
record:
- id: '14592'
relation: used_in_publication
status: public
- id: '12491'
relation: dissertation_contains
status: public
scopus_import: '1'
status: public
title: 3D printed cell culture grid holders for improved cellular specimen preparation
in cryo-electron microscopy
tmp:
image: /images/cc_by.png
legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
short: CC BY (4.0)
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 212
year: '2020'
...