---
_id: '13267'
abstract:
- lang: eng
text: Three-dimensional (3D) reconstruction of living brain tissue down to an individual
synapse level would create opportunities for decoding the dynamics and structure–function
relationships of the brain’s complex and dense information processing network;
however, this has been hindered by insufficient 3D resolution, inadequate signal-to-noise
ratio and prohibitive light burden in optical imaging, whereas electron microscopy
is inherently static. Here we solved these challenges by developing an integrated
optical/machine-learning technology, LIONESS (live information-optimized nanoscopy
enabling saturated segmentation). This leverages optical modifications to stimulated
emission depletion microscopy in comprehensively, extracellularly labeled tissue
and previous information on sample structure via machine learning to simultaneously
achieve isotropic super-resolution, high signal-to-noise ratio and compatibility
with living tissue. This allows dense deep-learning-based instance segmentation
and 3D reconstruction at a synapse level, incorporating molecular, activity and
morphodynamic information. LIONESS opens up avenues for studying the dynamic functional
(nano-)architecture of living brain tissue.
acknowledged_ssus:
- _id: ScienComp
- _id: Bio
- _id: PreCl
- _id: E-Lib
- _id: LifeSc
- _id: M-Shop
acknowledgement: "We thank J. Vorlaufer, N. Agudelo and A. Wartak for microscope maintenance
and troubleshooting, C. Kreuzinger and A. Freeman for technical assistance, M. Šuplata
for hardware control support and M. Cunha dos Santos for initial exploration of
software. We\r\nthank P. Henderson for advice on deep-learning training and M. Sixt,
S. Boyd and T. Weiss for discussions and critical reading of the manuscript. L.
Lavis (Janelia Research Campus) generously provided the JF585-HaloTag ligand. We
acknowledge expert support by IST\r\nAustria’s scientific computing, imaging and
optics, preclinical, library and laboratory support facilities and by the Miba machine
shop. We gratefully acknowledge funding by the following sources: Austrian Science
Fund (F.W.F.) grant no. I3600-B27 (J.G.D.), grant no. DK W1232\r\n(J.G.D. and J.M.M.)
and grant no. Z 312-B27, Wittgenstein award (P.J.); the Gesellschaft für Forschungsförderung
NÖ grant no. LSC18-022 (J.G.D.); an ISTA Interdisciplinary project grant (J.G.D.
and B.B.); the European Union’s Horizon 2020 research and innovation programme,\r\nMarie-Skłodowska
Curie grant 665385 (J.M.M. and J.L.); the European Union’s Horizon 2020 research
and innovation programme, European Research Council grant no. 715767, MATERIALIZABLE
(B.B.); grant no. 715508, REVERSEAUTISM (G.N.); grant no. 695568, SYNNOVATE (S.G.N.G.);
and grant no. 692692, GIANTSYN (P.J.); the Simons\r\nFoundation Autism Research
Initiative grant no. 529085 (S.G.N.G.); the Wellcome Trust Technology Development
grant no. 202932 (S.G.N.G.); the Marie Skłodowska-Curie Actions Individual Fellowship
no. 101026635 under the EU Horizon 2020 program (J.F.W.);\r\nthe Human Frontier
Science Program postdoctoral fellowship LT000557/2018 (W.J.); and the National Science
Foundation grant no. IIS-1835231 (H.P.) and NCS-FO-2124179 (H.P.)."
article_processing_charge: Yes
article_type: original
author:
- first_name: Philipp
full_name: Velicky, Philipp
id: 39BDC62C-F248-11E8-B48F-1D18A9856A87
last_name: Velicky
orcid: 0000-0002-2340-7431
- first_name: Eder
full_name: Miguel Villalba, Eder
id: 3FB91342-F248-11E8-B48F-1D18A9856A87
last_name: Miguel Villalba
orcid: 0000-0001-5665-0430
- first_name: Julia M
full_name: Michalska, Julia M
id: 443DB6DE-F248-11E8-B48F-1D18A9856A87
last_name: Michalska
orcid: 0000-0003-3862-1235
- first_name: Julia
full_name: Lyudchik, Julia
id: 46E28B80-F248-11E8-B48F-1D18A9856A87
last_name: Lyudchik
- first_name: Donglai
full_name: Wei, Donglai
last_name: Wei
- first_name: Zudi
full_name: Lin, Zudi
last_name: Lin
- first_name: Jake
full_name: Watson, Jake
id: 63836096-4690-11EA-BD4E-32803DDC885E
last_name: Watson
orcid: 0000-0002-8698-3823
- first_name: Jakob
full_name: Troidl, Jakob
last_name: Troidl
- first_name: Johanna
full_name: Beyer, Johanna
last_name: Beyer
- first_name: Yoav
full_name: Ben Simon, Yoav
id: 43DF3136-F248-11E8-B48F-1D18A9856A87
last_name: Ben Simon
- first_name: Christoph M
full_name: Sommer, Christoph M
id: 4DF26D8C-F248-11E8-B48F-1D18A9856A87
last_name: Sommer
orcid: 0000-0003-1216-9105
- first_name: Wiebke
full_name: Jahr, Wiebke
id: 425C1CE8-F248-11E8-B48F-1D18A9856A87
last_name: Jahr
- first_name: Alban
full_name: Cenameri, Alban
id: 9ac8f577-2357-11eb-997a-e566c5550886
last_name: Cenameri
- first_name: Johannes
full_name: Broichhagen, Johannes
last_name: Broichhagen
- first_name: Seth G.N.
full_name: Grant, Seth G.N.
last_name: Grant
- first_name: Peter M
full_name: Jonas, Peter M
id: 353C1B58-F248-11E8-B48F-1D18A9856A87
last_name: Jonas
orcid: 0000-0001-5001-4804
- first_name: Gaia
full_name: Novarino, Gaia
id: 3E57A680-F248-11E8-B48F-1D18A9856A87
last_name: Novarino
orcid: 0000-0002-7673-7178
- first_name: Hanspeter
full_name: Pfister, Hanspeter
last_name: Pfister
- first_name: Bernd
full_name: Bickel, Bernd
id: 49876194-F248-11E8-B48F-1D18A9856A87
last_name: Bickel
orcid: 0000-0001-6511-9385
- first_name: Johann G
full_name: Danzl, Johann G
id: 42EFD3B6-F248-11E8-B48F-1D18A9856A87
last_name: Danzl
orcid: 0000-0001-8559-3973
citation:
ama: Velicky P, Miguel Villalba E, Michalska JM, et al. Dense 4D nanoscale reconstruction
of living brain tissue. Nature Methods. 2023;20:1256-1265. doi:10.1038/s41592-023-01936-6
apa: Velicky, P., Miguel Villalba, E., Michalska, J. M., Lyudchik, J., Wei, D.,
Lin, Z., … Danzl, J. G. (2023). Dense 4D nanoscale reconstruction of living brain
tissue. Nature Methods. Springer Nature. https://doi.org/10.1038/s41592-023-01936-6
chicago: Velicky, Philipp, Eder Miguel Villalba, Julia M Michalska, Julia Lyudchik,
Donglai Wei, Zudi Lin, Jake Watson, et al. “Dense 4D Nanoscale Reconstruction
of Living Brain Tissue.” Nature Methods. Springer Nature, 2023. https://doi.org/10.1038/s41592-023-01936-6.
ieee: P. Velicky et al., “Dense 4D nanoscale reconstruction of living brain
tissue,” Nature Methods, vol. 20. Springer Nature, pp. 1256–1265, 2023.
ista: Velicky P, Miguel Villalba E, Michalska JM, Lyudchik J, Wei D, Lin Z, Watson
J, Troidl J, Beyer J, Ben Simon Y, Sommer CM, Jahr W, Cenameri A, Broichhagen
J, Grant SGN, Jonas PM, Novarino G, Pfister H, Bickel B, Danzl JG. 2023. Dense
4D nanoscale reconstruction of living brain tissue. Nature Methods. 20, 1256–1265.
mla: Velicky, Philipp, et al. “Dense 4D Nanoscale Reconstruction of Living Brain
Tissue.” Nature Methods, vol. 20, Springer Nature, 2023, pp. 1256–65, doi:10.1038/s41592-023-01936-6.
short: P. Velicky, E. Miguel Villalba, J.M. Michalska, J. Lyudchik, D. Wei, Z. Lin,
J. Watson, J. Troidl, J. Beyer, Y. Ben Simon, C.M. Sommer, W. Jahr, A. Cenameri,
J. Broichhagen, S.G.N. Grant, P.M. Jonas, G. Novarino, H. Pfister, B. Bickel,
J.G. Danzl, Nature Methods 20 (2023) 1256–1265.
date_created: 2023-07-23T22:01:13Z
date_published: 2023-08-01T00:00:00Z
date_updated: 2024-01-10T08:37:48Z
day: '01'
department:
- _id: PeJo
- _id: GaNo
- _id: BeBi
- _id: JoDa
- _id: Bio
doi: 10.1038/s41592-023-01936-6
ec_funded: 1
external_id:
isi:
- '001025621500001'
pmid:
- '37429995'
intvolume: ' 20'
isi: 1
language:
- iso: eng
main_file_link:
- open_access: '1'
url: https://doi.org/10.1038/s41592-023-01936-6
month: '08'
oa: 1
oa_version: Published Version
page: 1256-1265
pmid: 1
project:
- _id: 265CB4D0-B435-11E9-9278-68D0E5697425
call_identifier: FWF
grant_number: I03600
name: Optical control of synaptic function via adhesion molecules
- _id: 2548AE96-B435-11E9-9278-68D0E5697425
call_identifier: FWF
grant_number: W1232-B24
name: Molecular Drug Targets
- _id: 25C5A090-B435-11E9-9278-68D0E5697425
call_identifier: FWF
grant_number: Z00312
name: The Wittgenstein Prize
- _id: 23889792-32DE-11EA-91FC-C7463DDC885E
name: High content imaging to decode human immune cell interactions in health and
allergic disease
- _id: 2564DBCA-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '665385'
name: International IST Doctoral Program
- _id: 24F9549A-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '715767'
name: 'MATERIALIZABLE: Intelligent fabrication-oriented Computational Design and
Modeling'
- _id: 25444568-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '715508'
name: Probing the Reversibility of Autism Spectrum Disorders by Employing in vivo
and in vitro Models
- _id: 25B7EB9E-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '692692'
name: Biophysics and circuit function of a giant cortical glumatergic synapse
- _id: fc2be41b-9c52-11eb-aca3-faa90aa144e9
call_identifier: H2020
grant_number: '101026635'
name: Synaptic computations of the hippocampal CA3 circuitry
- _id: 2668BFA0-B435-11E9-9278-68D0E5697425
grant_number: LT00057
name: High-speed 3D-nanoscopy to study the role of adhesion during 3D cell migration
publication: Nature Methods
publication_identifier:
eissn:
- 1548-7105
issn:
- 1548-7091
publication_status: published
publisher: Springer Nature
quality_controlled: '1'
related_material:
link:
- relation: software
url: https://github.com/danzllab/LIONESS
record:
- id: '12817'
relation: research_data
status: public
- id: '14770'
relation: shorter_version
status: public
scopus_import: '1'
status: public
title: Dense 4D nanoscale reconstruction of living brain tissue
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 20
year: '2023'
...
---
_id: '11943'
abstract:
- lang: eng
text: Complex wiring between neurons underlies the information-processing network
enabling all brain functions, including cognition and memory. For understanding
how the network is structured, processes information, and changes over time, comprehensive
visualization of the architecture of living brain tissue with its cellular and
molecular components would open up major opportunities. However, electron microscopy
(EM) provides nanometre-scale resolution required for full in-silico
reconstruction1–5, yet is limited to fixed specimens and
static representations. Light microscopy allows live observation, with super-resolution
approaches6–12 facilitating nanoscale visualization, but
comprehensive 3D-reconstruction of living brain tissue has been hindered by tissue
photo-burden, photobleaching, insufficient 3D-resolution, and inadequate signal-to-noise
ratio (SNR). Here we demonstrate saturated reconstruction of living brain tissue.
We developed an integrated imaging and analysis technology, adapting stimulated
emission depletion (STED) microscopy6,13 in extracellularly
labelled tissue14 for high SNR and near-isotropic resolution.
Centrally, a two-stage deep-learning approach leveraged previously obtained information
on sample structure to drastically reduce photo-burden and enable automated volumetric
reconstruction down to single synapse level. Live reconstruction provides unbiased
analysis of tissue architecture across time in relation to functional activity
and targeted activation, and contextual understanding of molecular labelling.
This adoptable technology will facilitate novel insights into the dynamic functional
architecture of living brain tissue.
article_processing_charge: No
author:
- first_name: Philipp
full_name: Velicky, Philipp
id: 39BDC62C-F248-11E8-B48F-1D18A9856A87
last_name: Velicky
orcid: 0000-0002-2340-7431
- first_name: Eder
full_name: Miguel Villalba, Eder
id: 3FB91342-F248-11E8-B48F-1D18A9856A87
last_name: Miguel Villalba
orcid: 0000-0001-5665-0430
- first_name: Julia M
full_name: Michalska, Julia M
id: 443DB6DE-F248-11E8-B48F-1D18A9856A87
last_name: Michalska
orcid: 0000-0003-3862-1235
- first_name: Donglai
full_name: Wei, Donglai
last_name: Wei
- first_name: Zudi
full_name: Lin, Zudi
last_name: Lin
- first_name: Jake
full_name: Watson, Jake
id: 63836096-4690-11EA-BD4E-32803DDC885E
last_name: Watson
orcid: 0000-0002-8698-3823
- first_name: Jakob
full_name: Troidl, Jakob
last_name: Troidl
- first_name: Johanna
full_name: Beyer, Johanna
last_name: Beyer
- first_name: Yoav
full_name: Ben Simon, Yoav
id: 43DF3136-F248-11E8-B48F-1D18A9856A87
last_name: Ben Simon
- first_name: Christoph M
full_name: Sommer, Christoph M
id: 4DF26D8C-F248-11E8-B48F-1D18A9856A87
last_name: Sommer
orcid: 0000-0003-1216-9105
- first_name: Wiebke
full_name: Jahr, Wiebke
id: 425C1CE8-F248-11E8-B48F-1D18A9856A87
last_name: Jahr
- first_name: Alban
full_name: Cenameri, Alban
id: 9ac8f577-2357-11eb-997a-e566c5550886
last_name: Cenameri
- first_name: Johannes
full_name: Broichhagen, Johannes
last_name: Broichhagen
- first_name: Seth G. N.
full_name: Grant, Seth G. N.
last_name: Grant
- first_name: Peter M
full_name: Jonas, Peter M
id: 353C1B58-F248-11E8-B48F-1D18A9856A87
last_name: Jonas
orcid: 0000-0001-5001-4804
- first_name: Gaia
full_name: Novarino, Gaia
id: 3E57A680-F248-11E8-B48F-1D18A9856A87
last_name: Novarino
orcid: 0000-0002-7673-7178
- first_name: Hanspeter
full_name: Pfister, Hanspeter
last_name: Pfister
- first_name: Bernd
full_name: Bickel, Bernd
id: 49876194-F248-11E8-B48F-1D18A9856A87
last_name: Bickel
orcid: 0000-0001-6511-9385
- first_name: Johann G
full_name: Danzl, Johann G
id: 42EFD3B6-F248-11E8-B48F-1D18A9856A87
last_name: Danzl
orcid: 0000-0001-8559-3973
citation:
ama: Velicky P, Miguel Villalba E, Michalska JM, et al. Saturated reconstruction
of living brain tissue. bioRxiv. doi:10.1101/2022.03.16.484431
apa: Velicky, P., Miguel Villalba, E., Michalska, J. M., Wei, D., Lin, Z., Watson,
J., … Danzl, J. G. (n.d.). Saturated reconstruction of living brain tissue. bioRxiv.
Cold Spring Harbor Laboratory. https://doi.org/10.1101/2022.03.16.484431
chicago: Velicky, Philipp, Eder Miguel Villalba, Julia M Michalska, Donglai Wei,
Zudi Lin, Jake Watson, Jakob Troidl, et al. “Saturated Reconstruction of Living
Brain Tissue.” BioRxiv. Cold Spring Harbor Laboratory, n.d. https://doi.org/10.1101/2022.03.16.484431.
ieee: P. Velicky et al., “Saturated reconstruction of living brain tissue,”
bioRxiv. Cold Spring Harbor Laboratory.
ista: Velicky P, Miguel Villalba E, Michalska JM, Wei D, Lin Z, Watson J, Troidl
J, Beyer J, Ben Simon Y, Sommer CM, Jahr W, Cenameri A, Broichhagen J, Grant SGN,
Jonas PM, Novarino G, Pfister H, Bickel B, Danzl JG. Saturated reconstruction
of living brain tissue. bioRxiv, 10.1101/2022.03.16.484431.
mla: Velicky, Philipp, et al. “Saturated Reconstruction of Living Brain Tissue.”
BioRxiv, Cold Spring Harbor Laboratory, doi:10.1101/2022.03.16.484431.
short: P. Velicky, E. Miguel Villalba, J.M. Michalska, D. Wei, Z. Lin, J. Watson,
J. Troidl, J. Beyer, Y. Ben Simon, C.M. Sommer, W. Jahr, A. Cenameri, J. Broichhagen,
S.G.N. Grant, P.M. Jonas, G. Novarino, H. Pfister, B. Bickel, J.G. Danzl, BioRxiv
(n.d.).
date_created: 2022-08-23T11:07:59Z
date_published: 2022-05-09T00:00:00Z
date_updated: 2024-03-25T23:30:11Z
day: '09'
department:
- _id: PeJo
- _id: GaNo
- _id: BeBi
- _id: JoDa
doi: 10.1101/2022.03.16.484431
language:
- iso: eng
main_file_link:
- open_access: '1'
url: https://doi.org/10.1101/2022.03.16.484431
month: '05'
oa: 1
oa_version: Preprint
publication: bioRxiv
publication_status: submitted
publisher: Cold Spring Harbor Laboratory
related_material:
record:
- id: '12470'
relation: dissertation_contains
status: public
status: public
title: Saturated reconstruction of living brain tissue
type: preprint
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
year: '2022'
...
---
_id: '9258'
acknowledgement: 'We thank S. van der Walt and K. Marchuk for discussion during development.
This project was funded by Packard Fellowship and Chan Zuckerberg Biohub Investigator
Awards to L.W.; STROBE: A NSF Science and Technology Center; an NSF Graduate Research
Fellowship awarded to H.P.; a Berkeley Institute for Data Science/UCSF Bakar Computational
Health Sciences Institute Fellowship awarded to H.P. with support from the Koret
Foundation, the Gordon and Betty Moore Foundation, and the Alfred P. Sloan Foundation
to the University of California, Berkeley. K.W.E., B.L. and M.T. were funded by
the Chan Zuckerberg Initiative and NIH grant P41GM135019.'
article_processing_charge: No
article_type: letter_note
author:
- first_name: Henry
full_name: Pinkard, Henry
last_name: Pinkard
- first_name: Nico
full_name: Stuurman, Nico
last_name: Stuurman
- first_name: Ivan E.
full_name: Ivanov, Ivan E.
last_name: Ivanov
- first_name: Nicholas M.
full_name: Anthony, Nicholas M.
last_name: Anthony
- first_name: Wei
full_name: Ouyang, Wei
last_name: Ouyang
- first_name: Bin
full_name: Li, Bin
last_name: Li
- first_name: Bin
full_name: Yang, Bin
last_name: Yang
- first_name: Mark A.
full_name: Tsuchida, Mark A.
last_name: Tsuchida
- first_name: Bryant
full_name: Chhun, Bryant
last_name: Chhun
- first_name: Grace
full_name: Zhang, Grace
last_name: Zhang
- first_name: Ryan
full_name: Mei, Ryan
last_name: Mei
- first_name: Michael
full_name: Anderson, Michael
last_name: Anderson
- first_name: Douglas P.
full_name: Shepherd, Douglas P.
last_name: Shepherd
- first_name: Ian
full_name: Hunt-Isaak, Ian
last_name: Hunt-Isaak
- first_name: Raymond L.
full_name: Dunn, Raymond L.
last_name: Dunn
- first_name: Wiebke
full_name: Jahr, Wiebke
id: 425C1CE8-F248-11E8-B48F-1D18A9856A87
last_name: Jahr
- first_name: Saul
full_name: Kato, Saul
last_name: Kato
- first_name: Loïc A.
full_name: Royer, Loïc A.
last_name: Royer
- first_name: Jay R.
full_name: Thiagarajah, Jay R.
last_name: Thiagarajah
- first_name: Kevin W.
full_name: Eliceiri, Kevin W.
last_name: Eliceiri
- first_name: Emma
full_name: Lundberg, Emma
last_name: Lundberg
- first_name: Shalin B.
full_name: Mehta, Shalin B.
last_name: Mehta
- first_name: Laura
full_name: Waller, Laura
last_name: Waller
citation:
ama: 'Pinkard H, Stuurman N, Ivanov IE, et al. Pycro-Manager: Open-source software
for customized and reproducible microscope control. Nature Methods. 2021;18(3):226-228.
doi:10.1038/s41592-021-01087-6'
apa: 'Pinkard, H., Stuurman, N., Ivanov, I. E., Anthony, N. M., Ouyang, W., Li,
B., … Waller, L. (2021). Pycro-Manager: Open-source software for customized and
reproducible microscope control. Nature Methods. Springer Nature. https://doi.org/10.1038/s41592-021-01087-6'
chicago: 'Pinkard, Henry, Nico Stuurman, Ivan E. Ivanov, Nicholas M. Anthony, Wei
Ouyang, Bin Li, Bin Yang, et al. “Pycro-Manager: Open-Source Software for Customized
and Reproducible Microscope Control.” Nature Methods. Springer Nature,
2021. https://doi.org/10.1038/s41592-021-01087-6.'
ieee: 'H. Pinkard et al., “Pycro-Manager: Open-source software for customized
and reproducible microscope control,” Nature Methods, vol. 18, no. 3. Springer
Nature, pp. 226–228, 2021.'
ista: 'Pinkard H, Stuurman N, Ivanov IE, Anthony NM, Ouyang W, Li B, Yang B, Tsuchida
MA, Chhun B, Zhang G, Mei R, Anderson M, Shepherd DP, Hunt-Isaak I, Dunn RL, Jahr
W, Kato S, Royer LA, Thiagarajah JR, Eliceiri KW, Lundberg E, Mehta SB, Waller
L. 2021. Pycro-Manager: Open-source software for customized and reproducible microscope
control. Nature Methods. 18(3), 226–228.'
mla: 'Pinkard, Henry, et al. “Pycro-Manager: Open-Source Software for Customized
and Reproducible Microscope Control.” Nature Methods, vol. 18, no. 3, Springer
Nature, 2021, pp. 226–28, doi:10.1038/s41592-021-01087-6.'
short: H. Pinkard, N. Stuurman, I.E. Ivanov, N.M. Anthony, W. Ouyang, B. Li, B.
Yang, M.A. Tsuchida, B. Chhun, G. Zhang, R. Mei, M. Anderson, D.P. Shepherd, I.
Hunt-Isaak, R.L. Dunn, W. Jahr, S. Kato, L.A. Royer, J.R. Thiagarajah, K.W. Eliceiri,
E. Lundberg, S.B. Mehta, L. Waller, Nature Methods 18 (2021) 226–228.
date_created: 2021-03-21T23:01:20Z
date_published: 2021-03-01T00:00:00Z
date_updated: 2023-08-07T14:19:08Z
day: '01'
department:
- _id: JoDa
doi: 10.1038/s41592-021-01087-6
external_id:
isi:
- '000625600600007'
pmid:
- '33674797'
intvolume: ' 18'
isi: 1
issue: '3'
language:
- iso: eng
main_file_link:
- open_access: '1'
url: https://doi.org/10.1038/s41592-021-01087-6
month: '03'
oa: 1
oa_version: Published Version
page: 226-228
pmid: 1
publication: Nature Methods
publication_identifier:
eissn:
- 1548-7105
issn:
- 1548-7091
publication_status: published
publisher: Springer Nature
quality_controlled: '1'
scopus_import: '1'
status: public
title: 'Pycro-Manager: Open-source software for customized and reproducible microscope
control'
type: journal_article
user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 18
year: '2021'
...
---
_id: '6808'
abstract:
- lang: eng
text: Super-resolution fluorescence microscopy has become an important catalyst
for discovery in the life sciences. In STimulated Emission Depletion (STED) microscopy,
a pattern of light drives fluorophores from a signal-emitting on-state to a non-signalling
off-state. Only emitters residing in a sub-diffraction volume around an intensity
minimum are allowed to fluoresce, rendering them distinguishable from the nearby,
but dark fluorophores. STED routinely achieves resolution in the few tens of nanometers
range in biological samples and is suitable for live imaging. Here, we review
the working principle of STED and provide general guidelines for successful STED
imaging. The strive for ever higher resolution comes at the cost of increased
light burden. We discuss techniques to reduce light exposure and mitigate its
detrimental effects on the specimen. These include specialized illumination strategies
as well as protecting fluorophores from photobleaching mediated by high-intensity
STED light. This opens up the prospect of volumetric imaging in living cells and
tissues with diffraction-unlimited resolution in all three spatial dimensions.
article_processing_charge: No
article_type: original
author:
- first_name: Wiebke
full_name: Jahr, Wiebke
id: 425C1CE8-F248-11E8-B48F-1D18A9856A87
last_name: Jahr
- first_name: Philipp
full_name: Velicky, Philipp
id: 39BDC62C-F248-11E8-B48F-1D18A9856A87
last_name: Velicky
orcid: 0000-0002-2340-7431
- first_name: Johann G
full_name: Danzl, Johann G
id: 42EFD3B6-F248-11E8-B48F-1D18A9856A87
last_name: Danzl
orcid: 0000-0001-8559-3973
citation:
ama: Jahr W, Velicky P, Danzl JG. Strategies to maximize performance in STimulated
Emission Depletion (STED) nanoscopy of biological specimens. Methods. 2020;174(3):27-41.
doi:10.1016/j.ymeth.2019.07.019
apa: Jahr, W., Velicky, P., & Danzl, J. G. (2020). Strategies to maximize performance
in STimulated Emission Depletion (STED) nanoscopy of biological specimens. Methods.
Elsevier. https://doi.org/10.1016/j.ymeth.2019.07.019
chicago: Jahr, Wiebke, Philipp Velicky, and Johann G Danzl. “Strategies to Maximize
Performance in STimulated Emission Depletion (STED) Nanoscopy of Biological Specimens.”
Methods. Elsevier, 2020. https://doi.org/10.1016/j.ymeth.2019.07.019.
ieee: W. Jahr, P. Velicky, and J. G. Danzl, “Strategies to maximize performance
in STimulated Emission Depletion (STED) nanoscopy of biological specimens,” Methods,
vol. 174, no. 3. Elsevier, pp. 27–41, 2020.
ista: Jahr W, Velicky P, Danzl JG. 2020. Strategies to maximize performance in STimulated
Emission Depletion (STED) nanoscopy of biological specimens. Methods. 174(3),
27–41.
mla: Jahr, Wiebke, et al. “Strategies to Maximize Performance in STimulated Emission
Depletion (STED) Nanoscopy of Biological Specimens.” Methods, vol. 174,
no. 3, Elsevier, 2020, pp. 27–41, doi:10.1016/j.ymeth.2019.07.019.
short: W. Jahr, P. Velicky, J.G. Danzl, Methods 174 (2020) 27–41.
date_created: 2019-08-12T16:36:32Z
date_published: 2020-03-01T00:00:00Z
date_updated: 2023-08-17T13:59:57Z
day: '01'
department:
- _id: JoDa
doi: 10.1016/j.ymeth.2019.07.019
external_id:
isi:
- '000525860400005'
pmid:
- '31344404'
intvolume: ' 174'
isi: 1
issue: '3'
language:
- iso: eng
main_file_link:
- open_access: '1'
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7100895/
month: '03'
oa: 1
oa_version: Submitted Version
page: 27-41
pmid: 1
project:
- _id: 265CB4D0-B435-11E9-9278-68D0E5697425
call_identifier: FWF
grant_number: I03600
name: Optical control of synaptic function via adhesion molecules
- _id: 2668BFA0-B435-11E9-9278-68D0E5697425
grant_number: LT00057
name: High-speed 3D-nanoscopy to study the role of adhesion during 3D cell migration
publication: Methods
publication_identifier:
issn:
- 1046-2023
publication_status: published
publisher: Elsevier
quality_controlled: '1'
scopus_import: '1'
status: public
title: Strategies to maximize performance in STimulated Emission Depletion (STED)
nanoscopy of biological specimens
type: journal_article
user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 174
year: '2020'
...
---
_id: '6029'
abstract:
- lang: eng
text: Protein micropatterning has become an important tool for many biomedical applications
as well as in academic research. Current techniques that allow to reduce the feature
size of patterns below 1 μm are, however, often costly and require sophisticated
equipment. We present here a straightforward and convenient method to generate
highly condensed nanopatterns of proteins without the need for clean room facilities
or expensive equipment. Our approach is based on nanocontact printing and allows
for the fabrication of protein patterns with feature sizes of 80 nm and periodicities
down to 140 nm. This was made possible by the use of the material X-poly(dimethylsiloxane)
(X-PDMS) in a two-layer stamp layout for protein printing. In a proof of principle,
different proteins at various scales were printed and the pattern quality was
evaluated by atomic force microscopy (AFM) and super-resolution fluorescence microscopy.
article_number: '655'
article_processing_charge: No
author:
- first_name: Marco
full_name: Lindner, Marco
last_name: Lindner
- first_name: Aliz
full_name: Tresztenyak, Aliz
last_name: Tresztenyak
- first_name: Gergö
full_name: Fülöp, Gergö
last_name: Fülöp
- first_name: Wiebke
full_name: Jahr, Wiebke
id: 425C1CE8-F248-11E8-B48F-1D18A9856A87
last_name: Jahr
- first_name: Adrian
full_name: Prinz, Adrian
last_name: Prinz
- first_name: Iris
full_name: Prinz, Iris
last_name: Prinz
- first_name: Johann G
full_name: Danzl, Johann G
id: 42EFD3B6-F248-11E8-B48F-1D18A9856A87
last_name: Danzl
orcid: 0000-0001-8559-3973
- first_name: Gerhard J.
full_name: Schütz, Gerhard J.
last_name: Schütz
- first_name: Eva
full_name: Sevcsik, Eva
last_name: Sevcsik
citation:
ama: Lindner M, Tresztenyak A, Fülöp G, et al. A fast and simple contact printing
approach to generate 2D protein nanopatterns. Frontiers in Chemistry. 2019;6.
doi:10.3389/fchem.2018.00655
apa: Lindner, M., Tresztenyak, A., Fülöp, G., Jahr, W., Prinz, A., Prinz, I., …
Sevcsik, E. (2019). A fast and simple contact printing approach to generate 2D
protein nanopatterns. Frontiers in Chemistry. Frontiers Media S.A. https://doi.org/10.3389/fchem.2018.00655
chicago: Lindner, Marco, Aliz Tresztenyak, Gergö Fülöp, Wiebke Jahr, Adrian Prinz,
Iris Prinz, Johann G Danzl, Gerhard J. Schütz, and Eva Sevcsik. “A Fast and Simple
Contact Printing Approach to Generate 2D Protein Nanopatterns.” Frontiers in
Chemistry. Frontiers Media S.A., 2019. https://doi.org/10.3389/fchem.2018.00655.
ieee: M. Lindner et al., “A fast and simple contact printing approach to
generate 2D protein nanopatterns,” Frontiers in Chemistry, vol. 6. Frontiers
Media S.A., 2019.
ista: Lindner M, Tresztenyak A, Fülöp G, Jahr W, Prinz A, Prinz I, Danzl JG, Schütz
GJ, Sevcsik E. 2019. A fast and simple contact printing approach to generate 2D
protein nanopatterns. Frontiers in Chemistry. 6, 655.
mla: Lindner, Marco, et al. “A Fast and Simple Contact Printing Approach to Generate
2D Protein Nanopatterns.” Frontiers in Chemistry, vol. 6, 655, Frontiers
Media S.A., 2019, doi:10.3389/fchem.2018.00655.
short: M. Lindner, A. Tresztenyak, G. Fülöp, W. Jahr, A. Prinz, I. Prinz, J.G. Danzl,
G.J. Schütz, E. Sevcsik, Frontiers in Chemistry 6 (2019).
date_created: 2019-02-17T22:59:24Z
date_published: 2019-01-24T00:00:00Z
date_updated: 2023-08-24T14:45:38Z
day: '24'
ddc:
- '540'
department:
- _id: JoDa
doi: 10.3389/fchem.2018.00655
external_id:
isi:
- '000456718000001'
file:
- access_level: open_access
checksum: 7841301d7c53b56ef873791b4b6f7b24
content_type: application/pdf
creator: dernst
date_created: 2019-02-18T15:10:34Z
date_updated: 2020-07-14T12:47:17Z
file_id: '6039'
file_name: 2019_frontiers_Lindner.pdf
file_size: 1766820
relation: main_file
file_date_updated: 2020-07-14T12:47:17Z
has_accepted_license: '1'
intvolume: ' 6'
isi: 1
language:
- iso: eng
month: '01'
oa: 1
oa_version: Published Version
publication: Frontiers in Chemistry
publication_identifier:
eissn:
- '22962646'
publication_status: published
publisher: Frontiers Media S.A.
quality_controlled: '1'
scopus_import: '1'
status: public
title: A fast and simple contact printing approach to generate 2D protein nanopatterns
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: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 6
year: '2019'
...