Correlative light- and electron microscopy with chemical tags.
J Struct Biol
; 186(2): 205-13, 2014 May.
Article
em En
| MEDLINE
| ID: mdl-24698954
ABSTRACT
Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25 nm in the x-y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Coloração e Rotulagem
/
Microscopia Eletrônica
/
Corantes Fluorescentes
Limite:
Animals
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article