@article{6808,
  abstract     = {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.},
  author       = {Jahr, Wiebke and Velicky, Philipp and Danzl, Johann G},
  issn         = {1046-2023},
  journal      = {Methods},
  number       = {3},
  pages        = {27--41},
  publisher    = {Elsevier},
  title        = {{Strategies to maximize performance in STimulated Emission Depletion (STED) nanoscopy of biological specimens}},
  doi          = {10.1016/j.ymeth.2019.07.019},
  volume       = {174},
  year         = {2020},
}

@article{6059,
  abstract     = {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       = {Balta, Emre and Stopp, Julian A and Castelletti, Laura and Kirchgessner, Henning and Samstag, Yvonne and Wabnitz, Guido H.},
  issn         = {1046-2023},
  journal      = {Methods},
  number       = {1},
  pages        = {25--38},
  publisher    = {Elsevier},
  title        = {{Qualitative and quantitative analysis of PMN/T-cell interactions by InFlow and super-resolution microscopy}},
  doi          = {10.1016/j.ymeth.2016.09.013},
  volume       = {112},
  year         = {2017},
}