Details

Autor(en) / Beteiligte

• Jahr, Wiebke
• Velicky, Philipp
• Danzl, Johann Georg

Titel

Strategies to maximize performance in STimulated Emission Depletion (STED) nanoscopy of biological specimens

Ist Teil von

• Methods (San Diego, Calif.), 2020-03, Vol.174, p.27-41

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2020

Quelle

MEDLINE

Beschreibungen/Notizen

• •The concepts of super-resolution microscopy, and specifically STED, are explained.•The interaction of light with the samples is explained, with special focus on STED.•General laboratory guidelines for successful STED data acquisition are provided.•Recent developments to reduce light exposure and photobleaching are reviewed.•Technical developments to parallelize and speed up image acquisition are discussed. 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.