Strategies to maximize performance in STimulated Emission Depletion (STED) nanoscopy of biological specimens.
Methods
; 174: 27-41, 2020 03 01.
Article
de En
| MEDLINE
| ID: mdl-31344404
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.
Mots clés
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Traitement d'image par ordinateur
/
Microscopie de fluorescence
Langue:
En
Journal:
Methods
Sujet du journal:
BIOQUIMICA
Année:
2020
Type de document:
Article
Pays d'affiliation:
Autriche