Reversible cryo-arrest for imaging molecules in living cells at high spatial resolution.
Nat Methods
; 13(8): 665-672, 2016 08.
Article
em En
| MEDLINE
| ID: mdl-27400419
ABSTRACT
The dynamics of molecules in living cells hampers precise imaging of molecular patterns by functional and super-resolution microscopy. We developed a method that circumvents lethal chemical fixation and allows on-stage cryo-arrest for consecutive imaging of molecular patterns within the same living, but arrested, cells. The reversibility of consecutive cryo-arrests was demonstrated by the high survival rate of different cell lines and by intact growth factor signaling that was not perturbed by stress response. Reversible cryo-arrest was applied to study the evolution of ligand-induced receptor tyrosine kinase activation at different scales. The nanoscale clustering of epidermal growth factor receptor (EGFR) in the plasma membrane was assessed by single-molecule localization microscopy, and endosomal microscale activity patterns of ephrin receptor A2 (EphA2) were assessed by fluorescence lifetime imaging microscopy. Reversible cryo-arrest allows the precise determination of molecular patterns while conserving the dynamic capabilities of living cells.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Temperatura Baixa
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Crioprotetores
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Receptor EphA2
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Imagem Molecular
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Receptores ErbB
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Microscopia de Fluorescência
Limite:
Humans
Idioma:
En
Revista:
Nat Methods
Assunto da revista:
TECNICAS E PROCEDIMENTOS DE LABORATORIO
Ano de publicação:
2016
Tipo de documento:
Article
País de afiliação:
Alemanha