Cryo-planing of frozen-hydrated samples using cryo triple ion gun milling (CryoTIGM™).
J Struct Biol
; 192(3): 569-579, 2015 Dec.
Article
em En
| MEDLINE
| ID: mdl-26549007
ABSTRACT
Cryo-SEM is a high throughput technique for imaging biological ultrastructure in its most pristine state, i.e. without chemical fixation, embedding, or drying. Freeze fracture is routinely used to prepare internal surfaces for cryo-SEM imaging. However, the propagation of the fracture plane is highly dependent on sample properties, and the resulting surface frequently shows substantial topography, which can complicate image analysis and interpretation. We have developed a broad ion beam milling technique, called cryogenic triple ion gun milling (CryoTIGM™ ['kri-É-,tim]), for cryo-planing frozen-hydrated biological specimens. Comparing sample preparation by CryoTIGM™ and freeze fracture in three model systems, Baker's yeast, mouse liver tissue, and whole sea urchin embryos, we find that CryoTIGM™ yields very large (â¼700,000 µm(2)) and smooth sections that present ultrastructural details at similar or better quality than freeze-fractured samples. A particular strength of CryoTIGM™ is the ability to section samples with hard-soft contrast such as brittle calcite (CaCO3) spicules in the sea urchin embryo.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
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Hepatócitos
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Strongylocentrotus purpuratus
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Embrião não Mamífero
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Técnica de Fratura por Congelamento
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Fígado
Limite:
Animals
Idioma:
En
Revista:
J Struct Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2015
Tipo de documento:
Article