Single-molecule imaging of transcription factor binding to DNA in live mammalian cells.
Nat Methods
; 10(5): 421-6, 2013 May.
Article
em En
| MEDLINE
| ID: mdl-23524394
ABSTRACT
Imaging single fluorescent proteins in living mammalian cells is challenged by out-of-focus fluorescence excitation. To reduce out-of-focus fluorescence we developed reflected light-sheet microscopy (RLSM), a fluorescence microscopy method allowing selective plane illumination throughout the nuclei of living mammalian cells. A thin light sheet parallel to the imaging plane and close to the sample surface is generated by reflecting an elliptical laser beam incident from the top by 90° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to that in previous illumination schemes and enables imaging of single fluorescent proteins with up to 100-Hz time resolution. We demonstrated the single-molecule sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determining the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor-α (ER), which permitted us to resolve different modes of DNA binding of GR. We demonstrated two-color single-molecule imaging by observing the spatiotemporal colocalization of two different protein pairs. Our single-molecule measurements and statistical analysis revealed dynamic properties of transcription factors.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fatores de Transcrição
/
DNA
Limite:
Animals
Idioma:
En
Revista:
Nat Methods
Assunto da revista:
TECNICAS E PROCEDIMENTOS DE LABORATORIO
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Estados Unidos