Super-resolution microscopy using standard fluorescent proteins in intact cells under cryo-conditions.
Nano Lett
; 14(7): 4171-5, 2014 Jul 09.
Article
em En
| MEDLINE
| ID: mdl-24884378
ABSTRACT
We introduce a super-resolution technique for fluorescence cryo-microscopy based on photoswitching of standard genetically encoded fluorescent marker proteins in intact mammalian cells at low temperature (81 K). Given the limit imposed by the lack of cryo-immersion objectives, current applications of fluorescence cryo-microscopy to biological specimens achieve resolutions between 400-500 nm only. We demonstrate that the single molecule characteristics of reversible photobleaching of mEGFP and mVenus at liquid nitrogen temperature are suitable for the basic concept of single molecule localization microscopy. This enabled us to perform super-resolution imaging of vitrified biological samples and to visualize structures in unperturbed fast frozen cells for the first time with a structural resolution of â¼125 nm (average single molecule localization accuracy â¼40 nm), corresponding to a 3-5 fold resolution improvement.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Corantes Fluorescentes
/
Proteínas Luminescentes
/
Microscopia de Fluorescência
Limite:
Animals
Idioma:
En
Revista:
Nano Lett
Ano de publicação:
2014
Tipo de documento:
Article
País de afiliação:
Reino Unido