---
OA_place: publisher
OA_type: hybrid
_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 (in subscription journal)
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
  orcid: 0000-0003-0201-2315
- 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. <i>Nature Methods</i>. 2023;20:1256-1265. doi:<a href="https://doi.org/10.1038/s41592-023-01936-6">10.1038/s41592-023-01936-6</a>
  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. <i>Nature Methods</i>. Springer Nature. <a href="https://doi.org/10.1038/s41592-023-01936-6">https://doi.org/10.1038/s41592-023-01936-6</a>
  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.” <i>Nature Methods</i>. Springer Nature, 2023. <a href="https://doi.org/10.1038/s41592-023-01936-6">https://doi.org/10.1038/s41592-023-01936-6</a>.
  ieee: P. Velicky <i>et al.</i>, “Dense 4D nanoscale reconstruction of living brain
    tissue,” <i>Nature Methods</i>, 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.” <i>Nature Methods</i>, vol. 20, Springer Nature, 2023, pp. 1256–65, doi:<a
    href="https://doi.org/10.1038/s41592-023-01936-6">10.1038/s41592-023-01936-6</a>.
  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.
corr_author: '1'
date_created: 2023-07-23T22:01:13Z
date_published: 2023-08-01T00:00:00Z
date_updated: 2026-07-06T12:49:46Z
day: '01'
ddc:
- '570'
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'
file:
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has_accepted_license: '1'
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language:
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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
  name: Molecular Drug Targets
- _id: 25C5A090-B435-11E9-9278-68D0E5697425
  call_identifier: FWF
  grant_number: Z00312
  name: Synaptic communication in neuronal microcircuits
- _id: 23889792-32DE-11EA-91FC-C7463DDC885E
  grant_number: LS18-022
  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 glutamatergic 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
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    status: public
  - id: '11943'
    relation: earlier_version
    status: public
scopus_import: '1'
status: public
title: Dense 4D nanoscale reconstruction of living brain tissue
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: 20
year: '2023'
...