---
_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. <i>Methods</i>. 2020;174(3):27-41.
    doi:<a href="https://doi.org/10.1016/j.ymeth.2019.07.019">10.1016/j.ymeth.2019.07.019</a>
  apa: Jahr, W., Velicky, P., &#38; Danzl, J. G. (2020). Strategies to maximize performance
    in STimulated Emission Depletion (STED) nanoscopy of biological specimens. <i>Methods</i>.
    Elsevier. <a href="https://doi.org/10.1016/j.ymeth.2019.07.019">https://doi.org/10.1016/j.ymeth.2019.07.019</a>
  chicago: Jahr, Wiebke, Philipp Velicky, and Johann G Danzl. “Strategies to Maximize
    Performance in STimulated Emission Depletion (STED) Nanoscopy of Biological Specimens.”
    <i>Methods</i>. Elsevier, 2020. <a href="https://doi.org/10.1016/j.ymeth.2019.07.019">https://doi.org/10.1016/j.ymeth.2019.07.019</a>.
  ieee: W. Jahr, P. Velicky, and J. G. Danzl, “Strategies to maximize performance
    in STimulated Emission Depletion (STED) nanoscopy of biological specimens,” <i>Methods</i>,
    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.” <i>Methods</i>, vol. 174,
    no. 3, Elsevier, 2020, pp. 27–41, doi:<a href="https://doi.org/10.1016/j.ymeth.2019.07.019">10.1016/j.ymeth.2019.07.019</a>.
  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: '6059'
abstract:
- lang: eng
  text: Neutrophils or polymorphonuclear cells (PMN) eliminate bacteria via phagocytosis
    and/or NETosis. Apartfrom these conventional roles, PMN also have immune-regulatory
    functions. They can transdifferentiateand upregulate MHCII as well as ligands
    for costimulatory receptors which enables them to behave asantigen presenting
    cells (APC). The initial step for activating T-cells is the formation of an immunesynapse
    between T-cells and antigen-presenting cells. However, the immune synapse that
    develops atthe PMN/T-cell contact zone is as yet hardly investigated due to the
    non-availability of methods foranalysis of large number of PMN interactions. In
    order to overcome these obstacles, we introduce herea workflow to analyse the
    immune synapse of primary human PMN and T-cells using multispectral imag-ing flow
    cytometry (InFlow microscopy) and super-resolution microscopy. For that purpose,
    we used CD3and CD66b as the lineage markers for T-cells and PMN, respectively.
    Thereafter, we applied and criticallydiscussed various ‘‘masks” for identification
    of T-cell PMN interactions. Using this approach, we foundthat a small fraction
    of transdifferentiated PMN (CD66b+CD86high) formed stable PMN/T-cell conjugates.Interestingly,
    while both CD3 and CD66b accumulation in the immune synapse was dependent on thematuration
    state of the PMN, only CD3 accumulation was greatly enhanced by the presence of
    superanti-gen. The actin cytoskeleton was weakly rearranged at the PMN side on
    the immune synapse upon contactwith a T-cell in the presence of superantigen.
    A more detailed analysis using super-resolution microscopy(structured-illumination
    microscopy, SIM) confirmed this finding. Together, we present an InFlow micro-scopy
    based approach for the large scale analysis of PMN/T-cell interactions and – combined
    with SIM – apossibility for an in-depth analysis of protein translocation at the
    site of interactions.
author:
- first_name: Emre
  full_name: Balta, Emre
  last_name: Balta
- first_name: Julian A
  full_name: Stopp, Julian A
  id: 489E3F00-F248-11E8-B48F-1D18A9856A87
  last_name: Stopp
- first_name: Laura
  full_name: Castelletti, Laura
  last_name: Castelletti
- first_name: Henning
  full_name: Kirchgessner, Henning
  last_name: Kirchgessner
- first_name: Yvonne
  full_name: Samstag, Yvonne
  last_name: Samstag
- first_name: Guido H.
  full_name: Wabnitz, Guido H.
  last_name: Wabnitz
citation:
  ama: Balta E, Stopp JA, Castelletti L, Kirchgessner H, Samstag Y, Wabnitz GH. Qualitative
    and quantitative analysis of PMN/T-cell interactions by InFlow and super-resolution
    microscopy. <i>Methods</i>. 2017;112(1):25-38. doi:<a href="https://doi.org/10.1016/j.ymeth.2016.09.013">10.1016/j.ymeth.2016.09.013</a>
  apa: Balta, E., Stopp, J. A., Castelletti, L., Kirchgessner, H., Samstag, Y., &#38;
    Wabnitz, G. H. (2017). Qualitative and quantitative analysis of PMN/T-cell interactions
    by InFlow and super-resolution microscopy. <i>Methods</i>. Elsevier. <a href="https://doi.org/10.1016/j.ymeth.2016.09.013">https://doi.org/10.1016/j.ymeth.2016.09.013</a>
  chicago: Balta, Emre, Julian A Stopp, Laura Castelletti, Henning Kirchgessner, Yvonne
    Samstag, and Guido H. Wabnitz. “Qualitative and Quantitative Analysis of PMN/T-Cell
    Interactions by InFlow and Super-Resolution Microscopy.” <i>Methods</i>. Elsevier,
    2017. <a href="https://doi.org/10.1016/j.ymeth.2016.09.013">https://doi.org/10.1016/j.ymeth.2016.09.013</a>.
  ieee: E. Balta, J. A. Stopp, L. Castelletti, H. Kirchgessner, Y. Samstag, and G.
    H. Wabnitz, “Qualitative and quantitative analysis of PMN/T-cell interactions
    by InFlow and super-resolution microscopy,” <i>Methods</i>, vol. 112, no. 1. Elsevier,
    pp. 25–38, 2017.
  ista: Balta E, Stopp JA, Castelletti L, Kirchgessner H, Samstag Y, Wabnitz GH. 2017.
    Qualitative and quantitative analysis of PMN/T-cell interactions by InFlow and
    super-resolution microscopy. Methods. 112(1), 25–38.
  mla: Balta, Emre, et al. “Qualitative and Quantitative Analysis of PMN/T-Cell Interactions
    by InFlow and Super-Resolution Microscopy.” <i>Methods</i>, vol. 112, no. 1, Elsevier,
    2017, pp. 25–38, doi:<a href="https://doi.org/10.1016/j.ymeth.2016.09.013">10.1016/j.ymeth.2016.09.013</a>.
  short: E. Balta, J.A. Stopp, L. Castelletti, H. Kirchgessner, Y. Samstag, G.H. Wabnitz,
    Methods 112 (2017) 25–38.
date_created: 2019-02-26T13:45:17Z
date_published: 2017-01-01T00:00:00Z
date_updated: 2021-01-12T08:05:57Z
day: '01'
doi: 10.1016/j.ymeth.2016.09.013
extern: '1'
external_id:
  pmid:
  - '27693880'
intvolume: '       112'
issue: '1'
language:
- iso: eng
month: '01'
oa_version: None
page: 25-38
pmid: 1
publication: Methods
publication_identifier:
  issn:
  - 1046-2023
publication_status: published
publisher: Elsevier
quality_controlled: '1'
related_material:
  link:
  - relation: supplementary_material
    url: http://dx.doi.org/10.1016/j.ymeth.2016.09.013
status: public
title: Qualitative and quantitative analysis of PMN/T-cell interactions by InFlow
  and super-resolution microscopy
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 112
year: '2017'
...