Preparation of cryofixed cells for improved 3D ultrastructure with scanning transmission electron tomography.
Histochem Cell Biol
; 135(1): 1-9, 2011 Jan.
Article
em En
| MEDLINE
| ID: mdl-21113611
ABSTRACT
Scanning transmission electron tomography offers enhanced contrast compared to regular transmission electron microscopy, and thicker samples, up to 1 µm or more, can be analyzed, since the depth of focus and inelastic scattering are not limitations. In this study, we combine this novel imaging approach with state of the art specimen preparation by using novel light transparent sapphire specimen carrier for high-pressure freezing and a freeze substitution protocol for better contrast of membranes. This combination allows for imaging membranes and other subcellular structures with unsurpassed quality. This is demonstrated with mitochondria, where the inner and outer mitochondrial membranes as well as the membranes in the cristae appear in very close apposition with a minimal intermembrane space. These findings correspond well with old observations using freeze fracturing. In 880-nm thick sections of hemophagocytes, the three-dimensional structure of membrane sheets could be observed in the virtual sections of the tomogram. Microtubules, actin and intermediate filaments could be visualized within one sample. Intermediate filaments, however, could even be better observed in 3D using surface scanning electron tomography.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Citoesqueleto
/
Criopreservação
/
Tomografia com Microscopia Eletrônica
Limite:
Humans
Idioma:
En
Revista:
Histochem Cell Biol
Assunto da revista:
CITOLOGIA
/
HISTOCITOQUIMICA
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Alemanha