---
_id: '12567'
abstract:
- lang: eng
  text: Single-molecule localization microscopy (SMLM) greatly advances structural
    studies of diverse biological tissues. For example, presynaptic active zone (AZ)
    nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ
    proteins usually relies on epitope preservation using aldehyde-based immunocompetent
    fixation. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze
    substitution (FS), are widely used for ultrastructural studies of presynaptic
    architecture in electron microscopy (EM). HPF/FS demonstrated nearer-to-native
    preservation of AZ ultrastructure, e.g., by facilitating single filamentous structures.
    Here, we present a protocol combining the advantages of HPF/FS and direct stochastic
    optical reconstruction microscopy (dSTORM) to quantify nanotopology of the AZ
    scaffold protein Bruchpilot (Brp) at neuromuscular junctions (NMJs) of Drosophila
    melanogaster. Using this standardized model, we tested for preservation of Brp
    clusters in different FS protocols compared to classical aldehyde fixation. In
    HPF/FS samples, presynaptic boutons were structurally well preserved with ~22%
    smaller Brp clusters that allowed quantification of subcluster topology. In summary,
    we established a standardized near-to-native preparation and immunohistochemistry
    protocol for SMLM analyses of AZ protein clusters in a defined model synapse.
    Our protocol could be adapted to study protein arrangements at single-molecule
    resolution in other intact tissue preparations.
acknowledgement: This work has been supported by funding of the German Research Foundation
  (Deutsche Forschungsgemeinschaft [DFG], CRC 166, Project B06 to M.H. and A.-L.S.,
  FOR 3004 SYNABS P1 to M.H.) and by the Interdisciplinary Clinical Research Center
  (IZKF) Würzburg (Z-3/69 to M.M.P., N-229 to M.H. and A.-L.S.). A.M. is funded by
  the University of Leipzig Clinician Scientist Program.
article_number: '2128'
article_processing_charge: No
article_type: original
author:
- first_name: Achmed
  full_name: Mrestani, Achmed
  last_name: Mrestani
- first_name: Katharina
  full_name: Lichter, Katharina
  id: 39302e62-fcfc-11ec-8196-8b01447dbd3d
  last_name: Lichter
- first_name: Anna Leena
  full_name: Sirén, Anna Leena
  last_name: Sirén
- first_name: Manfred
  full_name: Heckmann, Manfred
  last_name: Heckmann
- first_name: Mila M.
  full_name: Paul, Mila M.
  last_name: Paul
- first_name: Martin
  full_name: Pauli, Martin
  last_name: Pauli
citation:
  ama: Mrestani A, Lichter K, Sirén AL, Heckmann M, Paul MM, Pauli M. Single-molecule
    localization microscopy of presynaptic active zones in Drosophila melanogaster
    after rapid cryofixation. <i>International Journal of Molecular Sciences</i>.
    2023;24(3). doi:<a href="https://doi.org/10.3390/ijms24032128">10.3390/ijms24032128</a>
  apa: Mrestani, A., Lichter, K., Sirén, A. L., Heckmann, M., Paul, M. M., &#38; Pauli,
    M. (2023). Single-molecule localization microscopy of presynaptic active zones
    in Drosophila melanogaster after rapid cryofixation. <i>International Journal
    of Molecular Sciences</i>. MDPI. <a href="https://doi.org/10.3390/ijms24032128">https://doi.org/10.3390/ijms24032128</a>
  chicago: Mrestani, Achmed, Katharina Lichter, Anna Leena Sirén, Manfred Heckmann,
    Mila M. Paul, and Martin Pauli. “Single-Molecule Localization Microscopy of Presynaptic
    Active Zones in Drosophila Melanogaster after Rapid Cryofixation.” <i>International
    Journal of Molecular Sciences</i>. MDPI, 2023. <a href="https://doi.org/10.3390/ijms24032128">https://doi.org/10.3390/ijms24032128</a>.
  ieee: A. Mrestani, K. Lichter, A. L. Sirén, M. Heckmann, M. M. Paul, and M. Pauli,
    “Single-molecule localization microscopy of presynaptic active zones in Drosophila
    melanogaster after rapid cryofixation,” <i>International Journal of Molecular
    Sciences</i>, vol. 24, no. 3. MDPI, 2023.
  ista: Mrestani A, Lichter K, Sirén AL, Heckmann M, Paul MM, Pauli M. 2023. Single-molecule
    localization microscopy of presynaptic active zones in Drosophila melanogaster
    after rapid cryofixation. International Journal of Molecular Sciences. 24(3),
    2128.
  mla: Mrestani, Achmed, et al. “Single-Molecule Localization Microscopy of Presynaptic
    Active Zones in Drosophila Melanogaster after Rapid Cryofixation.” <i>International
    Journal of Molecular Sciences</i>, vol. 24, no. 3, 2128, MDPI, 2023, doi:<a href="https://doi.org/10.3390/ijms24032128">10.3390/ijms24032128</a>.
  short: A. Mrestani, K. Lichter, A.L. Sirén, M. Heckmann, M.M. Paul, M. Pauli, International
    Journal of Molecular Sciences 24 (2023).
date_created: 2023-02-19T23:00:56Z
date_published: 2023-01-21T00:00:00Z
date_updated: 2023-08-01T13:16:36Z
day: '21'
ddc:
- '570'
department:
- _id: PeJo
doi: 10.3390/ijms24032128
external_id:
  isi:
  - '000930324700001'
file:
- access_level: open_access
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  date_created: 2023-02-20T07:09:27Z
  date_updated: 2023-02-20T07:09:27Z
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  file_name: 2023_IJMS_Mrestani.pdf
  file_size: 2823025
  relation: main_file
  success: 1
file_date_updated: 2023-02-20T07:09:27Z
has_accepted_license: '1'
intvolume: '        24'
isi: 1
issue: '3'
language:
- iso: eng
month: '01'
oa: 1
oa_version: Published Version
publication: International Journal of Molecular Sciences
publication_identifier:
  eissn:
  - 1422-0067
publication_status: published
publisher: MDPI
quality_controlled: '1'
scopus_import: '1'
status: public
title: Single-molecule localization microscopy of presynaptic active zones in Drosophila
  melanogaster after rapid cryofixation
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: 24
year: '2023'
...