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Nat Methods. 2013 May;10(5):421-6. doi: 10.1038/nmeth.2411. Epub 2013 Mar 24.

Single-molecule imaging of transcription factor binding to DNA in live mammalian cells.

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1
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.

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.

PMID:
23524394
PMCID:
PMC3664538
DOI:
10.1038/nmeth.2411
[Indexed for MEDLINE]
Free PMC Article
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