--- _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' ...