title: Single-molecule imaging of transcription factor binding to DNA in live mammalian cells.
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.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: J Christof M
authors.initials: JC
authors.lastname: Gebhardt
authors.forename: David M
authors.initials: DM
authors.lastname: Suter
authors.forename: Rahul
authors.initials: R
authors.lastname: Roy
authors.forename: Ziqing W
authors.initials: ZW
authors.lastname: Zhao
authors.forename: Alec R
authors.initials: AR
authors.lastname: Chapman
authors.forename: Srinjan
authors.initials: S
authors.lastname: Basu
authors.forename: Tom
authors.initials: T
authors.lastname: Maniatis
authors.forename: X Sunney
authors.initials: XS
authors.lastname: Xie
mesh_terms: D000818:Animals; D004247:DNA / Q000378:metabolism*; D008322:Mammals; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D014157:Transcription Factors; D004247:DNA