title: Fluorescence lifetime imaging of nuclear DNA: effect of fluorescence resonance energy transfer.
abstract: BACKGROUND
  DNA fluorescence dyes have been used to study DNA dynamics, chromatin structure, and cell cycle analysis. However, most microscopic fluorescence studies of DNA use only steady-state measurements and do not take advantage of the additional information content of the time-resolved fluorescence. In this paper, we combine fluorescence imaging of DNA with time-resolved measurements to examine the proximity of donors and acceptors bound to chromatin.
  
  
  METHODS
  We used frequency-domain fluorescence lifetime imaging microscopy to study the spatial distribution of DNA-bound donors and acceptors in fixed 3T3 nuclei. Over 50 cell nuclei were imaged in the presence of an AT-specific donor, Hoechst 33258 (Ho), and a GC-specific acceptor, 7-aminoactinomycin D (7-AAD).
  
  
  RESULTS
  The intensity images of Ho alone showed a spatially irregular distribution due to the various concentrations of DNA or AT-rich DNA throughout the nuclei. The lifetime imaging of the Ho-stained nuclei was typically flat. Addition of 7-AAD decreased the fluorescence intensity and lifetime of the Ho-stained DNA. The spatially dependent phase and modulation values of Ho in the presence of 7-AAD showed that the Ho decay becomes nonexponential, as is expected for a resonance energy transfer (RET) with multiple acceptors located over a range of distances. In approximately 40 nuclei, the intensity and lifetime decrease was spatially homogeneous. In approximately 10 nuclei, addition of 7-AAD resulted in a spatially nonhomogeneous decrease in intensity and lifetime. The RET efficiency was higher in G(2)/M than in G(0/1) phase cells.
  
  
  CONCLUSIONS
  Because RET efficiency depends on the average distance between Ho and 7-AAD, data suggest that the heterogeneity of lifetimes and spatial variation of the RET efficiency are caused by the presence of highly condensed regions of DNA in nuclei.
authors.affiliation: Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: S
authors.initials: S
authors.lastname: Murata
authors.forename: P
authors.initials: P
authors.lastname: Herman
authors.forename: H J
authors.initials: HJ
authors.lastname: Lin
authors.forename: J R
authors.initials: JR
authors.lastname: Lakowicz
mesh_terms: D016475:3T3 Cells; D020856:AT Rich Sequence / Q000187:drug effects; D000818:Animals; D006690:Bisbenzimidazole / Q000494:pharmacology; D002467:Cell Nucleus / Q000648:ultrastructure*; D002843:Chromatin / Q000648:ultrastructure*; D004247:DNA / Q000032:analysis* / Q000187:drug effects; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D020862:GC Rich Sequence / Q000187:drug effects; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D013050:Spectrometry, Fluorescence; D013996:Time and Motion Studies
chemical_list: D002843:Chromatin; D005456:Fluorescent Dyes; D003609:Dactinomycin; C025942:7-aminoactinomycin D; D004247:DNA; D006690:Bisbenzimidazole